Evidence for the common origin of viral and cellular sequences involved in sarcomagenic transformation.

The src genes of six different strains of avian sarcoma virus (ASV) were compared with those of a series of newly isolated sarcoma viruses, termed "recovery avian sarcoma viruses" (rASV's). The rASV's were isolated recently from chicken and quail tumors induced by transformation-defective (td) deletion mutants of Schmidt-Ruppin Rous sarcoma virus. The RNase T1-resistant oligonucleotide maps were constructed for the RNA genomes of different strains of ASV and td mutants. The src-specific sequences, characterized by RNase T1-resistant oligonucleotides ranging from 9 to 19 nucleotides long, were defined as those mapping between approximately 600 and 2,800 nucleotides from the 3' polyadenylate end of individual sarcoma viral RNAs, and missing in the corresponding td viral RNAs. Our results revealed that 12 src-specific oligonucleotides were highly conserved among several strains of ASV, including the rASV's, whereas certain strains of ASV were found to contain one to three characteristic src-specific oligonucleotides. We previously presented evidence supporting the idea that most of the src-specific sequences present in rASV RNAs are derived from cellular genetic information. Our present data indicate that the src genes of rASV's are closely related to other known ASVs. We conclude that the src genes of different strains of ASV and the cellular sarc sequences are of common origin, although some divergence has occurred among different viral src genes and related cellular sequences.     

Analysis of the src gene of sarcoma viruses generated by recombination between transformation-defective mutants and quail cellular sequences.

Tumors were produced in quails about 2 months after injection with a transformation-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (SR-A), that retains a small portion of the src gene. Sarcoma viruses were isolated from each of five such tumors. A transformation-defective mutant which has a nearly complete deletion of the src gene was unable to induce tumors. The avian sarcoma viruses recovered from quail tumors (rASV-Q) had biological properties similar to those of the avian sarcoma viruses previously acquired from chicken tumors (rASV-C); these chicken tumors had been induced by the same transformation-defective mutants. Both rASV-Q and rASV-C transformed cells in culture with similar focus morphology and produced tumors within 7 to 14 days after injection into chickens or quails. The size of rASV-Q genomic RNA was indistinguishable from that of SR-A by polyacrylamide gel electrophoresis. The sequences of rASV-Q RNA genomes were analyzed and compared with those of the parental transformation-defective virus, SR-A and of rASV-C by RNase T1 fingerprinting and oligonucleotide mapping. We found that the src sequences of all five isolates of rASV-Q were identical to each other but different from those of SR-A and rASV-C. Of 13 oligonucleotides of rASV-Q identified as src specific, two were not found in either SR-A or rASV-C RNA. Furthermore, some oligonucleotides present in SR-A or rASV-C or both were absent in rASV-Q. No differences were found for the sequences outside the src region in any of the viruses examined. In addition, rASV-Q-infected cells possessed a 60,000-dalton protein specifically precipitable by rabbit serum raised against SR-D-induced tumors. The facts that the src sequences are essentially the same for rASV's recovered from one animal species and different for rASV's obtained from different species provide conclusive evidence that cellular sequences of normal birds were inserted into the viral genome and supplied to the resulting recombinant viruses genetic information for cell transformation.     

Induction of tumors and generation of recovered sarcoma viruses by, and mapping of deletions in, two molecularly cloned src deletion mutants.

td108 , a transformation-defective (td) deletion mutant of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A (SR-A), was molecularly cloned. Two isolates of td viruses, td108 -3b and td108 -4a, obtained by transfection of the molecularly cloned td108 DNAs into chicken embryo fibroblasts, were tested for their ability to induce tumors and generate recovered avian sarcoma viruses ( rASVs ) in chickens. Both td viruses were able to induce tumors with a latency and frequency similar to those observed previously with biologically purified td mutants of SR-A. rASVs were isolated from most of the tumors examined. The genomic RNAs of those newly obtained rASVs were analyzed by RNase T1 oligonucleotide fingerprinting. The results showed that they had regained the deleted src sequences and contained the same set of marker src oligonucleotides as those of rASVs analyzed previously. The src oligonucleotides of rASVs are distinguishable from those present in SR-A. We conclude that those rASVs must have been generated by recombination between the molecularly cloned td mutants and the c-src sequence. The deletions in the td mutants were mapped by restriction enzyme analysis and nucleotide sequencing. td108 -3b was found to contain an internal src deletion of 1,416 nucleotides and to retain 57 and 105 nucleotides of the 5' and 3' src coding sequences, respectively. td108 -4a contained a src deletion of 1,174 nucleotides and retained 180 and 225 nucleotides of the 5' and 3' src sequences, respectively. Comparison of sequences in the 5' src and its upstream region of td108 -3b with those of SR-A, rASV1441 (a td108 -derived rASV analyzed previously), and c-src suggested that the 5' recombination between td108 and c-src occurred from 7 to 20 nucleotides upstream from the beginning of the src coding sequence.     

DNA sequence of the viral and cellular src gene of chickens. II. Comparison of the src genes of two strains of avian sarcoma virus and of the cellular homolog

The nucleotide sequence of the src gene and flanking regions of the Schmidt-Ruppin strain of Rous sarcoma virus (SR-A) was determined. The src region of SR-A was very homologous to that of recovered avian sarcoma virus (rASV1441), with only 17 differences among 1,578 nucleotides. The size of the predicted protein was 526 amino acids in both viruses, of which 6 amino acids were different. The differences in nucleotides and amino acids between the two viruses localized within the 5' two-thirds of the src coding region. There were also viruses localized within the 5' two-thirds of the src coding region. There were also some differences in the region flanking the 5' end of src. Since rASVs are considered to be recombinatns between deletion mutants of SR-A and cellular-src (c-src) sequences, several segments of c-src DNA were also sequenced to understand the molecular basis for the recombination. At 14 of 17 bases where SR-A and rASV1441 differed, rASV1441 had the same sequence as c-src. Three of these sequences corresponded to sequences of oligonucleotides which were previously identified in RNAs of nearly all isolates of rASV but which were absent in SR-A RNA. In the 5'-flanking sequences of the src gene, c-src was more similar to rASV1441 than to SR-A. These results confirm the cellular origin of the src sequences of rASVs and provide information about the possible sites of the recombination.     

Identification of the viral sequence required for the generation of recovered avian sarcoma viruses and characterization of a series of replication-defective recovered avian sarcoma viruses.

The ability of transformation-defective deletion mutants of Schmidt-Ruppin Rous sarcoma virus to induce tumors and generate recovered sarcoma viruses (rASVs) was correlated with the partial src sequences retained in the transformation-defective viral genomes. Since all the transformation-defective viruses that were capable of generating rASVs retained a portion of the 3' src sequence, regardless of the extent of the 5' src deletion, and those lacking the 3' src were unable to generate rASVs, it appears that the 3', but most likely not the 5', src sequence retained in the transformation-defective viral genome is essential for rASV formation. However, rASVs derived from a particular mutant, td109, which retained a portion of the 3' src sequence, but lacked most (if not all) of the 5' src sequence, were all found to be defective in replication. Analyses of the genomic sequences of 13 isolates of td109-derived rASVs revealed that they contained various deletions in viral envelope (env), polymerase (pol), and structural protein (gag) genes. Ten isolates of rASVs contained env deletions. One isolate (rASV3812) contained a deletion of env and the 3' half of pol, and one isolate (rASV398) contained a deletion of env and pol. The one with the most extensive deletion (rASV374) had a deletion from the p12-coding sequence through pol and env. In addition, the 5' src region of td109-derived rASVs were heterogeneous. Among the 7 isolates analyzed in detail, one isolate of rASV had a small deletion of the 5' src sequence, whereas three other isolates contained extra new sequences upstream from src. Both env- and env- pol- rASVs were capable of directing the synthesis of precursor and mature gag proteins in the infected nonproducer cells. We attribute the deletions in the replication-defective rASVs to the possibility that the 5' recombination site between the td109 and c-src sequence, involved regions of only partial homology due to lack of sufficient 5' src sequence in the td109 genome for homologous recombination. A model of recombination between the viral genome and the c-src sequence is proposed to account for the requirement of the 3' src sequence and the basis for the generation of deletions in td109-derived rASVs.     

Suppression of Rous sarcoma virus-induced tumor formation by preinfection with viruses encoding src protein with novel N termini.

Two recovered avian sarcoma viruses (rASVs), rASV157 and rASV1702, encode src products which contain novel, nonmyristoylated N-terminal amino acids. These viruses transform chicken embryo fibroblasts and cause tumors in chicks. However, the tumors rASVs induce are small and regress within 2 weeks. To determine whether this regression results from weak tumorigenicity or from the active immunity of the host, we injected 1-week-old chicks with rASV and several days later injected the chicks with challenge virus of a different subgroup. Of the rASV1702-preinfected chicks challenged 5 days later with Rous sarcoma virus (RSV), 40% showed no subsequent tumor formation and 60% formed tumors which regressed within 1 week. The potency of this protective effect depended on the dosage of preinfection virus used and increased as the interval between preinfection and challenge infection was lengthened (when the interval was 9 days, none of the challenged chicks formed tumors). rASV157-preinfected chicks challenged with RSV after 9 days showed only partial protection: 42% formed tumors which regressed, whereas 58% formed tumors which continued to grow. Challenging rASV-preinfected chicks with Fujinami sarcoma virus or a RSV vector encoding the v-fps oncogene or polyomavirus middle T resulted in no suppression of tumor formation. Preinfection with src mutants or a RSV vector encoding polyomavirus middle T antigen, both of which induce slow-growing tumors, failed to elicit the protective effect. Finally, a novel N-terminal domain encoded by rASV1702 src was shown to be involved in but not sufficient for full protection. These data indicate that determinants on or induced by rASV157 and rASV1702 can elicit a potent protection against the tumorigenic potential of RSV-encoded p60v-src.     

Transduction of c-src coding and intron sequences by a transformation-defective deletion mutant of Rous sarcoma virus.

The mechanism of cellular src (c-src) transduction by a transformation-defective deletion mutant, td109, of Rous sarcoma virus was studied by sequence analysis of the recombinational junctions in three td109-derived recovered sarcoma viruses (rASVs). Our results show that two rASVs have been generated by recombination between td109 and c-src at the region between exons 1 and 2 defined previously. Significant homology between td109 and c-src sequences was present at the sites of recombination. The viral and c-src sequence junction of the third rASV was formed by splicing a cryptic donor site at the 5' region of env of td109 to exon 1 of c-src. Various lengths of c-src internal intron 1 sequences were incorporated into all three rASV genomes, which resulted from activation of potential splice donor and acceptor sites. The incorporated intron 1 sequences were absent in the c-src mRNA, excluding its being the precursor for recombination with td109 and implying that initial recombinations most likely took place at the DNA level. A potential splice acceptor site within the incorporated intron 1 sequences in two rASVs was activated and was used for the src mRNA synthesis in infected cells. The normal env mRNA splice acceptor site was used for src mRNA synthesis for the third rASV.     

Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation.

We have shown previously that the membrane association of the src proteins of recovered avian sarcoma viruses (rASVs) 1702 (56 kilodaltons) and 157 (62.5 kilodaltons), whose size variations occur within 8 kilodaltons of the amino terminus, is salt sensitive and that, in isotonic salt, these src proteins fractionate as soluble cytoplasmic proteins. In contrast, wild-type Rous sarcoma virus pp60src behaves as an integral plasma membrane protein in cellular fractionation studies and shows prominent membrane interaction by immunofluorescence microscopy. In this study we have examined the distribution of these size-variant src proteins between free and complexed forms, their subcellular localization by immunofluorescence microscopy, and their ability to effect several transformation-related cell properties. Glycerol gradient sedimentation of extracts from cells infected either with rASV 1702 or rASV 157 showed that soluble src proteins of these viruses were distributed between free and complexed forms as has been demonstrated for wild-type Rous sarcoma virus pp60src. Pulse-chase studies with rASV pp60src showed that, like wild-type Rous sarcoma virus pp60src, it was transiently found in a complexed form. Indirect immunofluorescence showed that size-variant pp60src proteins are localized in adhesion plaques and regions of cell-to-cell contact in rASV 1702- or 157-infected cells. This result is in contrast with the generalized localization of pp60src in plasma membranes of control rASV-infected cells which produce pp60src. Chicken embryo fibroblasts infected by rASVs 1702 and 157 display a partial-transformation phenotype with respect to (i) transformation-related morphology, (ii) cell surface membrane changes, and (iii) retained extracellular fibronectin. It is possible that the induction of a partial-transformation phenotype may be the result of the unique interaction of the src proteins encoded by these viruses with restricted areas of the plasma membrane.     

Heterogeneous RNA of the Prague strain of Rous sarcoma virus caused by undiluted passages.

The size of genomic RNA in PR-RSV A passaged in chick embryo fibroblasts (CEF) or quail embryo fibroblasts (QEF) was determined by gel electrophoresis. The results showed that 3 undiluted passages resulted in heterogeneity of RNA. The heterogeneity of the smaller incomplete RNAs in the virus stock was decreased by diluted passage or cloning, but RNA of the b subunit size and a subunit RNA of complete genome size were relatively stable. These heterogeneous RNAs were characterized by hybridization analysis. The RNAs from 4 peaks hybridized with both cDNAtotal and cDNAsrc to appreciable extents, indicating that they were derived from viral RNA and that at least some of them contained the src sequence. This finding and the failure to isolate a td mutant from the undiluted-passaged virus stock or from some subclones that had a and b subunits of RNA indicate that the td virus was only a minor constituent of the incomplete virus population caused by undiluted passages. Some viruses with incomplete RNA in the virus stock could produce foci with the aid of td B77 or RAV-60. The emergence of rd viruses by undiluted passages was indicated.     

Transformation-defective mutants of Rous sarcoma virus with src gene deletions of varying length.

The RNAs of transformation-defective (td) deletion mutants of the Schmidt-Ruppin strain of Rous sarcoma virus were found to vary in size when compared by polyacrylamide gel electrophoresis. Three of seven td mutants appeared to recombine with a mutant of Rous sarcoma virus (Schmidt-Ruppin), which has a temperature-sensitive sarcoma (src) gene and is termed ts68, to give rise to recombinants with a reduced temperature sensitivity. The results suggested that different clones of td mutants exist: some in which the src gene appears to be deleted, and others in which the src gene is only partially deleted. A direct correlation between RNA size and the extent of src gene deletion measured by recombination was not obtained, possibly because the recombination assay could only detect src sequences homologous to the lesion(s) of ts68, whereas the electrophoretic analysis of the RNA measured src deletions as well as other possible alterations of the RNA.     

Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity

We have suggested previously that the amino-terminal 8 kilodaltons of pp60^src may serve as a structural hydrophobic domain through which pp60^src attaches to plasma membranes. Two isolates of recovered avian sarcoma viruses (rASVs), 1702 and 157, encode pp60^src proteins that have alterations in this amino-terminal region. The rASV 1702 src protein (56 kilodaltons) and the 157 src protein (62.5 kilodaltons) show altered membrane association, and fractionate largely as soluble, cytoplasmic proteins in aqueous buffers, in contrast with the membrane association of more than 80% of the src protein of standard avian sarcoma virus under the identical fractionation procedure. Plasma membranes purified from cells transformed by these rASVs contain less than 10% of the amount of pp60^src found in membranes purified from cells transformed by Rous sarcoma virus or control rASVs. The altered membrane association of these src proteins had little or no effect on the properties of chick embryo fibroblasts transformed in monolayer culture. In contrast, rASV 1702 showed reduced in vivo tumorigenicity compared with Rous sarcoma virus or with other rASVs that encode membrane-associated src proteins. Rous sarcoma virus-induced tumors are malignant, poorly differentiated sarcomas that are lethal to their hosts. rASV 1702 induces a benign, differentiated sarcoma that regresses and is not lethal to its hosts. These data support the role of amino-terminal sequences in the membrane association of pp60^src, and suggest that the amino terminus of pp60^src may have a critical role in the promotion of in vivo tumorigenicity.     

Purification of DNA complementary to nucleotide sequences required for neoplastic transformation of fibroblasts by avian sarcoma viruses.

We have prepared radioactive DNA (cDNA_sarc) complementary to nucleotide sequences which represent at least a portion of the viral gene(s) required for neoplastic transformation of fibroblasts by an avian sarcoma virus. The genetic complexity of cDNA_sarc (~1600 nucleotides) is sufficient to represent an entire cistron. The genomes of three independent isolates of avian sarcoma viruses share nucleotide sequences closely related to cDNA_sarc, whereas the sequences are absent from transformation-defective mutants of avian sarcoma viruses, several avian leukosis viruses, a non-pathogenic endogenous virus of chickens (Rous-associated virus-O), sarcoma-leukosis viruses of mice and cats, and mouse mammary tumor virus. We conclude that the transforming gene(s) of all avian sarcoma viruses have closely related or common genetic lineages distinct from the transforming genes in sarcoma viruses of other species. Our results conform to previous reports that transformation-defective variants of avian sarcoma viruses are mutants with identical regions deleted from each subunit of a polyploid genome.     

Intracellular precursors to the major glycoprotein of avian oncoviruses in chicken embryo fibroblasts.

A 96,000-dalton glycoprotein, p(96), was present in cell extracts obtained from gs-chf- chicken embryo fibroblasts infected with the avian RNA tumor viruses Rous-associated virus-2 subgroup B (RAV-2) and the Schmidt-Ruppin strain of Rous sarcoma virus subgroup A (SR-RSV-A), as well as from uninfected gsLchf+ (HE) cell extracts. It was not found in cell extracts from uninfected gs-chf- or gs+chf+ (HH) cells, nor from gs-chf- cells infected with envelope-deficient Bryan high-titer Rous sarcoma virus. Immunoprecipitation, kinetic, and biochemical data indicate the this polyprotein contains information that gives rise to the major virion glycoprotein gp85. A second polyprotein of 80,000 daltons, p/80), is also present in the RAV-2- and SR-RSV-A-infected gs-chf- cells. This second polyprotein contains less carbohydrate than p(96), and kinetic and biochemical data indicate that p(80) may be an immature form of p(96).     

Transformation-defective mutants of Rous sarcoma virus with longer sizes of genome RNA and their highly frequent occurrences.

Transformation-defective (td) mutants with different sizes of genomic RNA were isolated from the Prague strain of Rous sarcoma virus, subgroup C(PR-C). All six td viruses (tdTYPR-C) isolated from a single UV-irradiated stock of PR-C (clone 2 of TYPR-C) had slightly longer RNA than did the ordinary class b RNA of tdB77 and Rous-associated virus-7. td viruses spontaneously segregated in uncloned TYPR-C also contained genomic RNA of a size similar to tdTYPR-C RNA. On the other hand, two td mutants isolated from another stock of PR-C (LAPR-C) had the class b RNA. Fingerprint analysis confirmed that tdTYPR-C and tdLAPR-C were derived by deletion from clone 2 of TYPR-C and LAPR-C, respectively, and also showed that clone 2 of TYPR-C had sequences in its genome RNA different from those of LAPR-C, although it gave a fingerprinting pattern similar to the latter. These results strongly suggest that differences between the nucleotide sequences in TYPR-C and LAPR-C RNA may result in different extents of deletion.     

Mapping RNase T1-resistant oligonucleotides of avian tumor virus RNAs: sarcoma-specific oligonucleotides are near the poly(A) end and oligonucleotides common to sarcoma and transformation-defective viruses are at the poly(A) end.

The large RNase T1-resistant oligonucleotides of the nondefective (nd) Rous sarcoma virus (RSV): Prague RSV of subgroup B (PR-B), PR-C and B77 of subgroup C; of their transformation-defective (td0 deletion mutants: td PR-B, td PR-C, and td B77; and of replication-defective (rd) RSV(-) were completely or partially mapped on the 30 to 40S viral RNAs. The location of a given oligonucleotide relative to the poly(A) terminus of the viral RNAs was directly deduced from the smallest size of the poly(A)-tagged RNA fragment from which it could be isolated. Identification of distinct oligonucleotides was based on their location in the electrophoretic/chromatographic fingerprint pattern and on analysis of their RNase A-resistant fragments. The following results were obtained. (i) The number of large oligonucleotides per poly(A)-tagged ffagment increased with increasing size of the fragment. This implies that the genetic map is linear and that a given RNase T1-resistant oligonucleotides has, relative to the poly(A) end, the same location on all 30 to 40S RNA subunits of a given 60 to 70S viral RNA complex, (ii) Three sarcoma-specific oligonucleotides were identified in the RNAs of Pr-B, PR-C and B77 by comparison with the RNAs of the corresponding td viruses...     

Nucleotide Sequence Relationships of Avian RNA Tumor Viruses: Measurement of the Deletion in a Transformation-Defective Mutant of Rous Sarcoma Virus

Stocks of cloned helper-independent Rous sarcoma virus (RSV) spontaneously segregate transformation-defective (td) mutants that appear to have an RNA genome composed of smaller subunits than those of the patent virus. Differential hybridization and competitive hybridization techniques involving reactions between viral RNA and proviral sequences in host cell DNA (under conditions of initial DNA excess) were used to measure the extent of the deletion in a td mutant of Prague strain (Pr) of RSV (Pr RSV-C). Viral 60 to 70S RNA sequences labeled to 1 to 5 x 10(7) counts per min per mug with (125)I were characterized with respect to their properties in hybridization reactions and used to reinforce data obtained with [(3)H]RNA of lower specific activity. By these techniques, about 13% +/- 3% of the sequences Pr RSV-C that formed hybrids with DNA from virus-induced sarcomas appeared to be deleted from the genome of td Pr RSV-C. Studies comparing hybridization of RNA from Pr RSV-C and td Pr RSV-C with RSV-related sequences in normal cells, and competition experiments with RNA from the endogenous chicken oncornavirus Rous-associated virus type 0 (RAV-0) provided evidence that the majority, if not all, of the RNA sequences of Pr RSV-C deleted from its transformation-defective mutant are not represented in normal chicken DNA. Competition studies with a leukosis virus, RAV-7, indicated this virus also lacks a genome segment of about the same size as the deletion in the td mutant. Finally, the genome of all three "exogenous" viruses was found to lack a small segment (about 12%) of sequences present in the endogenous provirus of RAV-O.     

Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.

Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes.     

Transformation-defective virus mutants in a class of morphologic revertants of sarcoma virus transformed nonproducer cells

In studies of the viral and cellular functions involved in expression of transformation by murine sarcoma virus, selective methods have led to the isolation of morphologic revertants following mitomycin C mutagenization of nonproductively transformed mouse cells. The revertants exhibit normal growth properties, yet still contain the sarcoma virus. Further, they are as susceptible as normal cells to exogenous sarcoma virus infection. In the present studies, these revertants are shown to contain levels of sarcoma viral RNA quantitatively and qualitatively indistinguishable from that present in the parental transformed clone. Following rescue with helper leukemia virus, they release low levels of wild-type transforming virus and a large excess of transformation-defective sarcoma virus as measured by molecular hybridization. The defective viruses can be transmitted to new cells in the absence of morphologic alteration. These results provide strong evidence that the revertants contain mutant viruses defective in transforming functions. The release of wild-type sarcoma virus by cells in a revertant culture appears to occur concomitantly with the spontaneous appearance of retransformed cells. This suggests that the reversion of mutant virus to wild-type within the cell occurs as a result of reversion of a point mutation in the integrated sarcoma viral genome. The present sarcoma virus mutants appear to be the first obtained by spontaneous or chemically-induced genetic alteration of stably integrated virus in eucaryotic cells.     

Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses.

The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that determines the host range and surface antigenicitiy of virions. We have purified radioactive DNA (cDNAgp) complementary to at least a portion of the env gene for viral subgroups A and C; complementary DNA was synthesized with purified virions of wild-type avian sarcoma virus, and RNA from a mutant with a deletion in env was used to select DNA specific to env by molecular hybridization. The genetic complexity of cDNAgp for subgroup A (ca. 2,000 nucleotides) was sufficient to represent the entire deletion and most or all of the env cistron. The deletions in env in two independently isolated strains of virus (Bryan and rdNY8SR) overlap, and cDNAgp represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletions in the adjacent viral gene src. Laboratory stocks of viral subgroups A, B, C, D and E do not contain detectable amounts of env deletions when tested by molecular hybridization; hence, segregation of deletions in env is a less frequent event that the segregation of deletions in the viral transforming gene src (Vogt, 1971). We found extensive homology among the nucleotide sequences encoding the env genes of virus strains indigenous to chickens (subgroups A, B, C, D, and E) although subgorups B, D and E appear to differ slightly from subgroups A and C at the env locus. By contrast, viruses obtained from pheasant cells (subgroups F and G) have env genes with little or no relationship to env genes of chikcen viruses. According to available data, viruses of subgroup F arose by recombination between an avarian sarcoma virus and viral genes in the genome of ring-necked pheasants, whereas subgroup G viruses may be entirely endogenous to golden pheasants.