Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA.

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was termed "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After cells acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplifications to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transformed, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.     

Molecular cloning, characterization, and genetic mapping of an endogenous murine mammary tumor virus proviral unit I of C3H/He mice.

We have cloned and characterized a novel endogenous murine mammary tumor virus proviral unit of the C3H/He strain of mice. The cloned proviral unit is 16 kilobase pairs (kbp) in size and is composed of a 5.6-kbp 5' EcoRI segment of an endogenous provirus with 10.4-kbp flanking cellular sequences. A comparison of the restriction map of the cloned proviral DNA with that of an endogenous provirus of the GR strain of mice has revealed minor differences in restriction sites on the two proviruses. The restriction enzyme SstI, which does not cleave the 5' EcoRI fragment of GR DNA, cleaves the C3H/He proviral sequences once; MspI has an additional site in the C3H/He proviral sequences. By using a subcloned fragment containing unique cellular sequences as a hybridization probe, we (i) mapped the C3H/He proviral unit to chromosome 14 by using mouse-hamster somatic cell hybrids, and (ii) demonstrated that this proviral unit is also present in the genome of DBA/2 mice. From these results we conclude that the C3H/He strain of mice acquired this proviral unit from DBA stock by genetic transmission. Our data also indicate that the murine mammary tumor virus sequences present in the gag-specific proviral unit of C3H/He mice extend at least 2.45 kbp downstream of the EcoRI site in the genomic DNA. Since the structural organization and chromosomal location of this proviral unit are distinct from those of previously reported proviral units represented by similar-sized (16.7-kbp) EcoRI fragments, we tentatively propose to designate this proviral unit Mtv-7a.     

McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (v-fms).

The genetic structure of the McDonough strain of feline sarcoma virus (SM-FeSV) was deduced by analysis of molecularly cloned, transforming proviral DNA. The 8.2-kilobase pair SM-FeSV provirus is longer than those of other feline sarcoma viruses and contains a transforming gene (v-fms) flanked by sequences derived from feline leukemia virus. The order of genes with respect to viral RNA is 5'-gag-fms-env-3', in which the entire feline leukemia virus env gene and an almost complete gag sequence are represented. Transfection of NIH/3T3 cells with cloned SM-FeSV proviral DNA induced foci of morphologically transformed cells which expressed SM-FeSV gene products and contained rescuable sarcoma viral genomes. Cells transformed by viral infection or after transfection with cloned proviral DNA expressed the polyprotein (P170gag-fms) characteristic of the SM-FeSV strain. Two proteolytic cleavage products (P120fms and pp55gag) were also found in immunoprecipitates from metabolically labeled, transformed cells. An additional polypeptide, detected at comparatively low levels in SM-FeSV transformants, was indistinguishable in size and antigenicity from the envelope precursor (gPr85env) of feline leukemia virus. The complexity of the v-fms gene (3.1 +/- 0.3 kilobase pairs) is approximately twofold greater than the viral oncogene sequences (v-fes) of Snyder-Theilen and Gardner-Arnstein FeSV. By heteroduplex, restriction enzyme, and nucleic acid hybridization analyses, v-fms and v-fes sequences showed no detectable homology to one another. Radiolabeled DNA fragments representing portions of the two viral oncogenes hybridized to different EcoRI and HindIII fragments of normal cat cellular DNA. Cellular sequences related to v-fms (designated c-fms) were much more complex than c-fes and were distributed segmentally over more than 40 kilobase pairs in cat DNA. Comparative structural studies of the molecularly cloned proviruses of Synder-Theilen, Gardner-Arnstein, and SM-FeSV showed that a region of the feline-leukemia virus genome derived from the pol-env junction is represented adjacent to v-onc sequences in each FeSV strain and may have provided sequences preferred for recombination with cellular genes.     

Transfection of Fibroblasts by Cloned Abelson Murine Leukemia Virus DNA and Recovery of Transmissible Virus by Recombination with Helper Virus

A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 10(4) transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3' end of the genome was not present. The transforming gene was thus localized to the 5' portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3' ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.     

Structural organization and biological activity of molecular clones of the integrated genome of a BALB/c mouse sarcoma virus.

BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin. The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda. In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol. Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection. A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome. Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA. A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA. A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs. These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.     

Biochemical characterization of cells transformed via transfection by feline sarcoma virus proviral DNA.

Murine fibroblasts transformed by transfection with DNA from mink cells infected with the Snyder-Theilen strain of feline sarcoma virus and subgroup B feline leukemia virus were analyzed for the presence of integrated proviral DNA and the expression of feline leukemia virus- and feline sarcoma virus-specific proteins. The transformed murine cells harbored at least one intact feline sarcoma virus provirus, but did not contain feline leukemia virus provirus. The transformed murine cells expressed an 85,000-dalton protein that was precipitated by antisera directed against feline leukemia virus p12, p15, and p30 proteins. No feline oncornavirus-associated cell membrane antigen reactivity was detected on the surfaces of the transformed murine cells by indirect membrane immunofluorescence techniques. The 85,000-dalton feline sarcoma virus-specific protein was also found in feline cells transformed by transfection. However, these cells also contained env gene products. The results of this study demonstrate that the feline sarcoma virus genome is sufficient to transform murine cells and that expression of the 85,000-dalton gag-x protein is associated with transformation of both murine and feline cells transformed by transfection.     

Subgenomic fragment of molecular cloned Friend murine leukemia virus DNA contains the gene(s) responsible for Friend murine leukemia virus-induced disease.

Friend murine leukemia virus (G-MuLV) is a helper-independent, type C retrovirus isolated from stocks of Friend virus complex (spleen focus-forming virus plus MuLV). In cell culture, F-MuLV has an ecotropic and NB-tropic host range and causes XC cells to fuse. When injected into newborn NIH Swiss mice, F-MuLV produces hepatosplenomegaly, severe anemia, and numerous circulating hematopoietic precursors in the peripheral blood with normal thymus and lymph nodes after 3 to 6 weeks. Recently, we molecularly cloned an 8.5-kilobase pair (kbp) form of F-MuLV DNA from which we could recover the pathogenic F-MuLV virus by DNA transfection of NIH 3T3 cells. From this molecularly cloned F-MuLV DNA, we have now subcloned in pBR322 a 4.1-kbp HindIII fragment which contains in continuity 3.0 kbp from the 3' terminus (env and c region), 0.6 kbp of the terminal repeat sequences, and 0.5 kbp from the 5'terminus of the viral RNA (genome). NIH 3T3 fibroblasts were transfected with this DNA fragment an then infected with the wild mouse amphotropic retrovirus (cl 1504-A). In cell culture, 1504-A is a helper-independent type C virus which has an N-tropic host range and does not cause fusion of XC cells. When injected into newborn NIH Swiss mice, 1504-A does not produce splenomegaly or thymic enlargement in mice held for up to 8 months. The transfection with the F-MuLV fragment and the infection with 1504-A consistently yielded virus preparations that were XC positive. From such virus stocks we were able to isolate both helper-independent and replication-defective XC-positive viruses. The helper-independent virus was shown to be a recombinant virus since it contains a gp70 molecule derived at least in part from F-MuLV and a specific gag precursor derived from 1504-A as determined by radioactive immune precipitation assays. When injected into newborn Swiss mice, the recombinant helper-independent virus caused hepatosplenomegaly in approximately 50% of the mice in 6 to 8 weeks. The histology of the diseased splenic tissue was indistinguishable from that seen in the disease caused by the whole F-MuLV. The replication-defective virus could be pseudotyped with new 1504-A virus, and this viral complex also caused the F-MuLV disease picture when the complex was injected into newborn Swiss mice. We conclude that the genetic information responsible for the pathogenicity of F-MuLV is contained within the 4.1-kbp DNA fragment, which includes env gene sequences, the terminal repeat sequences, and the c region sequences of the F-MuLV genome.     

Identification of the avian myeloblastosis virus genome. II. Restriction endonuclease analysis of DNA from lambda proviral recombinants and leukemic myeoblast clones.

Two lambda proviral DNA recombinants were characterized with a number of restriction endonucleases. One recombinant contained a complete presumptive avian myeloblastosis virus (AMV) provirus flanked by cellular sequences on either side, and the second recombinant contained 85% of a myeloblastosis-associated virus type 1 (MAV-1)-like provirus with cellular sequences adjacent to the 5' end of the provirus. Comparing the restriction maps for the proviral DNAs contained in each lambda hybrid showed that the putative AMV and MAV-1-like genomes shared identical enzyme sites for 3.6 megadaltons beginning at the 5' termini of the proviruses with respect to viral RNA. Two enzyme sites near the 3'-end of the MAV-1-like provirus were not present in the putative AMV genome. We also examined a number of leukemic myeloblast clones for proviral content and cell-provirus integration sites. The presumptive AMV provirus was present in all the leukemic myeloblast clones regardless of the endogenous proviral content of the target cells or the AMV pseudotype used for conversion. Multiple cellular sites were suitable for integration of the putative AMV genome and the helper genomes. The proviral genomes were all integrated colinearly with respect to linear viral DNA.     

Molecular cloning of bovine leukemia virus DNA integrated into the bovine tumor cell genome

The bovine leukemia virus (BLV) DNA harbored in the bovine tumor cell genome was cloned in lambda Charon 4A phage. Using either representative or 3' half-enriched BLV cDNA as a blot hybridization probe, clone lambda BLV-1 was shown to carry 9 kb of the BLV genome, flanked by cellular sequences at both ends. Restriction mapping with twelve endonucleases and hybridization of the DNA fragments to BLV cDNA representing a 3'-end portion of the viral genome revealed the presence and precise location of two long terminal repeats (LTRs) and virus-cell junctions. Thus, lambda BLV-1 appears to contain the complete BLV genome and flanking tumor cellular sequences. The restriction map of the cloned BLV proviral DNA closely resembles that previously reported for unintegrated linear proviral DNA, but differs significantly from that of the integrated provirus of another BLV isolate, the difference occurring preferentially in the putative gag and pol genes.     

Molecular cloning of the feline c-fes proto-oncogene and construction of a chimeric transforming gene

The feline c-fes proto-oncogene, different parts of which were captured in feline leukemia virus (FeLV) to generate the transforming genes (v-fes) of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) and the Snyder-Theilen strain (ST) of FeSV, was cloned and its genetic organization determined. Southern blot analysis revealed that the c-fes genetic sequences were distributed discontinuously and colinearly with the v-fes transforming gene over a DNA region of around 12.0 kb. Using cloned c-fes sequences, complementation of GA-FeSV transforming activity was studied. Upon replacement of the 3' half of v-fesGA with homologous feline c-fes sequences and transfection of the chimeric gene, morphological transformation was observed. Immunoprecipitation analysis of these transformed cells revealed expression of high Mr fusion proteins. Phosphorylation of these proteins was observed in an in vitro protein kinase assay, and tyrosine residues appeared to be involved as acceptor amino acid.     

In vitro methylation of specific regions of the cloned Moloney sarcoma virus genome inhibits its transforming activity.

The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as methylation at HpaII sites. In addition, methylation at both HpaII and HhaI sites did not further reduce the transforming activity of the DNA. These results suggested that; whereas methylation of specific sites on the provirus may not be essential for inhibiting the transforming activity of MSV DNA, methylation of specific regions may be necessary. Thus, by cotransfection of plasmids containing only specific regions of the MSV provirus, it was determined that methylation of the v-mos gene was more inhibitory to transformation than methylation of the viral long terminal repeat.     

Nature and distribution of feline sarcoma virus nucleotide sequences.

The genomes of three independent isolates of feline sarcoma virus (FeSV) were compared by molecular hybridization techniques. Using complementary DNAs prepared from two strains, SM- and ST-FeSV, common complementary DNA'S were selected by sequential hybridization to FeSV and feline leukemia virus RNAs. These DNAs were shown to be highly related among the three independent sarcoma virus isolates. FeSV-specific complementary DNAs were prepared by selection for hybridization by the homologous FeSV RNA and against hybridization by fline leukemia virus RNA. Sarcoma virus-specific sequences of SM-FeSV were shown to differ from those of either ST- or GA-FeSV strains, whereas ST-FeSV-specific DNA shared extensive sequence homology with GA-FeSV. By molecular hybridization, each set of FeSV-specific sequences was demonstrated to be present in normal cat cellular DNA in approximately one copy per haploid genome and was conserved throughout Felidae. In contrast, FeSV-common sequences were present in multiple DNA copies and were found only in Mediterranean cats. The present results are consistent with the concept that each FeSV strain has arisen by a mechanism involving recombination between feline leukemia virus and cat cellular DNA sequences, the latter represented within the cat genome in a manner analogous to that of a cellular gene.     

Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene.

The nucleotide sequence of the feline c-fes/fps proto-oncogene was analyzed. Comparison with v-fes and v-fps revealed that all v-fes/fps homologous sequences were dispersed over 11 kilobase pairs in 19 interspersed segments. All segments, numbered exon 1 to exon 19 as in the chicken and human loci, were flanked by consensus splice junctions. The putative promoter region contained a CATT sequence and three CCGCCC motifs which were also found in the human locus at similar positions. About 200 nucleotides downstream of a translational stop codon in exon 19, a putative poly(A) addition signal was identified. Using the putative translation initiation codon in exon 2, a 93,000-molecular-weight protein could be deduced. This protein resembled very well the putative protein of the human c-fes/fps proto-oncogene (94% overall homology) and, although less well, the putative protein of the chicken c-fes/fps proto-oncogene (70% overall homology). As far as the feline c-fes/fps proto-oncogene sequences transduced to the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) are concerned, homology in deduced amino acid sequences between the GA- and ST-v-fes viral oncogenes and the proto-oncogene was 99%. Analysis of the recombination junctions between feline leukemia virus and v-fes sequences in GA- and ST-FeSV proviral DNA revealed for the left-hand junction the involvement of homologous recombination, presumably at the DNA level. The right-hand junction, which appeared identical in the GA-FeSV and ST-FeSV genomes, could have been the result of a site-specific recombination at the RNA level.     

A new acute transforming feline retrovirus with fms homology specifies a C-terminally truncated version of the c-fms protein that is different from SM-feline sarcoma virus v-fms protein.

The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isolated from a multicentric fibrosarcoma of a domestic cat. The HZ5-FeSV transforms fibroblasts in vitro and is replication defective. A biologically active integrated HZ5-FeSV provirus was molecularly cloned from cellular DNA of HZ5-FeSV-infected FRE-3A rat cells. The HZ5-FeSV has oncogene homology with the fms sequences of the SM-FeSV. The genome organization of the 8.6-kilobase HZ5-FeSV provirus is 5' delta gag-fms-delta pol-delta env 3'. The HZ5-and SM-FeSVs display indistinguishable in vitro transformation characteristics, and the structures of the gag-fms transforming genes in the two viruses are very similar. In the HZ5-FeSV and the SM-FeSV, identical c-fms and feline leukemia virus p10 sequences form the 5' gag-fms junction. With regard to v-fms the two viruses are homologous up to 11 amino acids before the C terminus of the SM-FeSV v-fms protein. In HZ5-FeSV a segment of 362 nucleotides then follows before the 3' recombination site with feline leukemia virus pol. The new 3' v-fms sequence encodes 27 amino acids before reaching a TGA termination signal. The relationship of this sequence with the recently characterized human c-fms sequence has been examined. The 3' HZ5-FeSV v-fms sequence is homologous with 3' c-fms sequences. A frameshift mutation (11-base-pair deletion) was found in the C-terminal fms coding sequence of the HZ5-FeSV. As a result, the HZ5-FeSV v-fms protein is predicted to be a C-terminally truncated version of c-fms. This frameshift mutation may determine the oncogenic properties of v-fms in the HZ5-FeSV.     

Molecular cloning of integrated Gardner-Rasheed feline sarcoma virus: genetic structure of its cell-derived sequence differs from that of other tyrosine kinase-coding onc genes.

Gardner-Rasheed feline sarcoma virus (GR-FeSV) is an acute transforming retrovirus which encodes a gag-onc polyprotein possessing an associated tyrosine kinase activity. The integrated form of this virus, isolated in the Charon 21A strain of bacteriophage lambda, demonstrated an ability to transform NIH/3T3 cells at high efficiency upon transfection. Foci induced by GR-FeSV DNA contained rescuable sarcoma virus and expressed GR-P70, the major GR-FeSV translational product. The localization of long-terminal repeats within the DNA clone made it possible to establish the length of the GR-FeSV provirus as 4.6 kilobase pairs. The analysis of heteroduplexes formed between lambda feline leukemia virus (FeLV) and lambda GR-FeSV DNAs revealed the presence of a 1,700-base-pair FeLV unrelated segment, designated v-fgr, within the GR-FeSV genome. The size of this region was sufficient to encode a protein of approximately 68,000 daltons and was localized immediately downstream of the FeLV gag gene coding sequences present in GR-FeSV. Thus, it is likely that this 1.7-kilobase-pair stretch encodes the onc moiety of GR-P70. Utilizing probes representing v-fgr, we detected homologous sequences in the DNAs of diverse vertebrate species, implying that v-fgr originated from a well-conserved cellular gene. The number of cellular DNA fragments hybridized by v-fgr-derived probes indicated either that proto-fgr is distributed over a very large region of cellular DNA or represents a family of related genes. By molecular hybridization, v-fgr was not directly related to the onc genes of other known retroviruses having associated tyrosine kinase activity.     

Two unrelated cell-derived sequences in the genome of avian leukemia and carcinoma inducing retrovirus MH2.

Molecularly cloned proviral DNA of avian replication-defective retrovirus Mill Hill No. 2 (MH2) was analyzed. The MH2 provirus measures 5.5 kb including two long terminal repeats (LTR), and contains a partial complement of the structural gene gag, 1.5 kb in size, near the 5' terminus, and a 1.3-kb segment of the v-myc transforming gene near the 3' terminus. These v-myc sequences are closely related to the v-myc transforming gene of avian acute leukemia virus MC29, and to the cellular chicken gene c-myc. The gag and myc domains on the MH2 provirus are separated by unique sequences, 1.3 kb in size and termed v-mil, which are unrelated to v-myc, or to other oncogenes or structural genes of the avian leukemia-sarcoma group of retroviruses. Normal chicken DNA contains sequences closely related to v-mil, termed c-mil. Analyses of chicken c-mil clones isolated from a recombinant DNA library of the chicken genome reveal that c-mil is a single genetic locus with a complex split gene structure. In the MH2 genome, v-mil is expressed via genome-sized mRNA as a gag-related hybrid protein, p100gag-mil, while v-myc is apparently expressed via subgenomic mRNA independently from major coding regions of structural genes. The presence in the MH2 genome of two unrelated cell-derived sequences and their independent expression may be significant for the oncogenic specificities of this virus.     

Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus

Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.