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Several cDNA clones representing alternatively spliced Rev-specific transcripts were isolated from a cDNA library prepared from Himalayan tahr cells infected with caprine arthritis encephalitis virus (CAEV). We previously characterized two rev-like cDNA species, d1 and d2, and a tat e1 cDNA containing the rev coding sequence downstream to the tat. In these cDNAs, the rev coding domain derives its amino terminus from the N terminus of env, which is spliced to the 3' open reading frame encoding the putative Rev protein. In this study, we report the genetic structure of a fourth rev-like cDNA (designated g1), which lacks the 5' env-derived sequences. All of these rev transcripts, including cDNA g1, increased the level of chloramphenicol acetyltransferase expression when cotransfected with a reporter plasmid containing the CAEV Rev-response element-spanning region downstream of the cat coding sequences. Western blot (immunoblot) analysis showed that each transfected cDNA species gave rise to a 16-kDa protein lacking env-encoded amino-terminal epitopes. In contrast, CAEV-infected Himalayan tahr cells expressed only a 20-kDa protein, whose N terminus, in contrast, is derived from the env. Moreover, only the 20-kDa protein was also detected in the mature CAEV virions. These observations suggest that the transcripts d1, d2, and e1 can potentially, in appropriate cellular context, encode two Rev isoforms differing in their N termini, whereas the g1 transcript encodes only the 16-kDa species. Elucidation of the significance of the 16-kDa Rev protein in CAEV biology must await further studies.

Germline mutations in BRCA1 are responsible for most cases of inherited breast and ovarian cancer. However, the function of the BRCA1 protein has remained elusive. We now show that BRCA1 encodes a 190-kD protein with sequence homology and biochemical analogy to the granin protein family. Interestingly, BRCA2 also includes a motif similar to the granin consensus at the C terminus of the protein. Both BRCA1 and the granins localize to secretory vesicles, are secreted by a regulated pathway, are post-translationally glycosylated and are responsive to hormones. As a regulated secretory protein, BRCA1 appears to function by a mechanism not previously described for tumour suppressor gene products.

Colorectal cancer (CRC) is one of the most frequent cancers in humans. It develops via a multistage process involving alterations of both protooncogenes and tumor suppressor genes. In the present report we determined the level of expression of several Wnt genes in CRC by RT-PCR and direct sequencing. While Wnt-1 was not detectably expressed in any colonic tissues, Wnt-5a gene was efficiently expressed both in nontumorous as well as in colonic tumor tissues. In contrast, the Wnt-2 gene, which was expressed at low levels in normal colon, exhibited overexpression in all tumor tissue samples at the different Dukes' stages of CRC progression, including premalignant polyps and liver metastases. Overexpression of the Wnt-2 gene occurred also in other digestive neoplasms such as gastric and esophageal carcinomas, as well as in diverticulitis associated with stenosis or pseudo-tumor.

We have previously reported the tissue specific distribution of four different FGF-1 transcripts containing alternative 5' untranslated exons spliced to the first protein coding exon. The predominant transcript in brain is FGF-1.B and in kidney FGF-1.A. Others have shown, by in situ hybridization and immunohistochemical analysis, that expression of FGF-1 in the brain is exclusively in neural cells but not in glial cells. Here we have examined the distribution of FGF-1.B and FGF-1.A transcripts in glioblastoma and retinal tissues and in kidney carcinoma cell lines. Our results show that FGF-1.B is the predominant transcript in neural derived tissues including both the diabetic retina and normal retina tissues. Surprisingly, FGF-1.B transcript is highly expressed in glioblastoma tissues. In contrast, a normal brain glial cell line, CHII, expresses very low levels of FGF-1 mRNA. These results strongly implicate the role of FGF-1 in the etiology of glioblastoma. We also examined several kidney carcinoma derived cell lines for the expression of FGF-1 mRNA. Most of these kidney cell lines do not express any FGF-1 transcripts. An interpretation by deduction is that kidney adenocarcinomas are derived from cortex but medulla has been reported as the site of FGF-1 synthesis. Of the kidney derived cell lines which are positive for FGF-1 message, only one expressed FGF-1.A transcript. The data may suggest that the establishment of kidney cell lines results in a switch of promoter usage from the 1.A seen in kidney tissue. Similarly, culturing of glioma cell lines may result in a switch from FGF-1.B seen in glioma tissues to FGF-1.D seen in most glioma cell lines. Continued studies of the FGF-1 transcripts, their functional promoters and their tissues distribution will provide insight into the potential role of FGF-1 in cell growth, tissue differentiation and malignant transformation.

We analyzed the long terminal repeat (LTR) of the lymphoproliferative disease virus of turkeys for sequences that influence its promoter activity by using the chloramphenicol acetyltransferase assay. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 regulatory sequences were used for these studies. Through transfection experiments, we identified a negative regulatory element residing at the 5' end of the U3. The two imperfect direct repeats (DRs) located at nt - 170 to - 125 upstream of the RNA transcription site were identified as enhancer elements which could stimulate transcription of a heterologous promoter in an orientation independent manner. Specific interaction of nuclear factors with the DRs element was identified. The two DRs contain cArg motifs which are suggested to play a role in tissue specific expression of several cellular genes.

The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10(11) bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by 'scrape loading'. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.

Two distinct species of caprine arthritis encephalitis virus (CAEV) tat cDNAs were isolated early after infection of a Himalayan tahr cell line. Sequence analyses predicted that one cDNA (pCEV/e1) represented a polycistronic transcript that encodes Tat and Rev as well as an N-terminally truncated transmembrane protein and a protein, designated X, whose function is unknown; whereas the other cDNA (pCEV/f1) encodes Tat and the env gene products. pCEV/e1 trans-activated a CAEV LTR-chloramphenicol acetyltransferase reporter gene in goat synovial membrane cells. This activity was shown to be encoded by the Tat open reading frame by analysis of a deletion mutant. Because the pCAEV/f1 insert was unstable in plasmid form, its Tat activity could not be convincingly demonstrated. The target sequences for Tat within the CAEV LTR were localized to the U3 region which, when placed in either orientation upstream of heterologous promoters, was able to confer responsiveness to Tat.

The lymphoproliferative disease retrovirus (LPDV) induces an acute, horizontally transmitted disease of turkeys that is often fatal. Although LPDV cannot be grown in cultured cells, it was possible to isolate molecular clones of biologically active integrated proviral genomes from spleens of infected turkeys. Based upon molecular hybridization and nucleotide sequence comparisons of its pol gene, LPDV was shown to represent a distinct group of avian retroviruses most closely related to avian sarcoma-leukemia viruses. Here we report the complete nucleotide sequence of the LPDV genome as well as amino acid sequence analysis of its gag gene products. The genetic organization of LPDV is characteristic of members of the oncovirus subfamily. Further sequence comparisons of the gag gene confirmed that LPDV is most closely related to Rous sarcoma virus (RSV). However, the gag, pro, and pol open reading frames (ORFs) were in different translational phases so that the expression of their mature gene products would require the double frame-shifting mechanism utilized by simian retroviruses, mouse mammary tumor virus, and human T-cell leukemia virus. In contrast, the RSV proteinase is synthesized as part of the gag precursor. The LPDV gag gene differs from that of RSV as well as from all other retroviruses in that it encodes a unique 31,000-Da (p31) protein, located between the MA and the CA coding sequences. FOur short ORFs of unknown function were present, Whether the putative products of these ORFs account for the acute nature of LPDV-induced disease remains to be determined.

Lymphoproliferative disease virus, a type-C retrovirus, is the etiologic agent of a naturally acquired lymphoproliferative disorder in turkeys. The disease is characterized by rapid induction of lymphoproliferative lesions with morphologic and histopathologic features resembling those of reticuloendotheliosis. Owing to lack of overt clinical manifestations, early detection of lymphoproliferative disease virus is essential for preventing the rapid horizontal spread of the disease that can decimate flocks. We describe development of a simple, rapid, sensitive, and highly specific assay, using the polymerase chain reaction, capable of providing differential diagnosis of the disease soon after infection.

Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.

Three cDNA clones representing structurally distinct transcripts were isolated from a cDNA library prepared from cells infected with equine infectious anemia virus (EIAV) by using a probe representing the S3 open reading frame, which is thought to encode Rev. One species, designated p2/2, contained four exons and was identical to a previously described polycistronic mRNA that encodes Tat. This transcript was predicted to also direct the synthesis of a truncated form of the transmembrane protein and a putative Rev protein whose N-terminal 29 amino acids, derived from env, are linked to S3 sequences. The second cDNA, p176, also consisted of four exons which were generated by two of three of the same splicing events that occur with p2/2 but not with the Tat mRNA. The alternative splice site giving rise to the second exon of p176 results in a bicistronic message that would encode the same transmembrane and Rev proteins as p2/2. The first exon of the third transcript, p20, was identical to those of p2/2 and p176 but was spliced directly to S3. This monocistronic message could encode a second form of Rev that lacks env sequences, provided that Rev synthesis would initiate at a non-AUG codon. The coding capacity of each cDNA was assessed in a eukaryotic system using S3 antisera. Two putative Rev proteins with apparent molecular masses of 18 and 16 kDa were expressed by p2/2 and p176, while p20 expressed only a 16-kDa species. Analysis of EIAV-infected cells with S3 antisera revealed the presence of an 18-kDa protein. Surprisingly, the same protein was detected in purified virions. By using a reporter construct, the chloramphenicol acetyltransferase gene linked to EIAV env sequences, we were able to demonstrate greatly enhanced chloramphenicol acetyltransferase activity in cells cotransfected with this construct and any of the three cDNAs.

A cDNA predicted to encode a transmembrane tyrosine kinase receptor with sequence features characteristic of known fibroblast growth factor (FGF) receptors was isolated from an expression library constructed from the human mammary epithelial cell line B5/589. This cDNA, designated cl44, encodes a product of 803 amino acid residues and was readily distinguishable from known FGF receptors. During the course of our studies, Partanen et al. (Partanen, J., Makela, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson, W. L., and Alitalo, K. (1991) EMBO J. 10, 1347-1354) isolated a new FGF receptor, designated FGFR4, from the human leukemia cell line, K562. Its amino acid sequence is identical to that of cl44 with the exception of 1 residue. The 5'-untranslated sequences of the two cDNAs diverged far upstream of the initiation codon. A myoblast line, L6E9, which lacks FGF receptors, was utilized to express high levels of FGFR4. We found, in contrast to Partenen et al., who reported only binding of acidic FGF, that FGFR4 bound both acidic and basic FGF with dissociation constants of 10-15 and 120 pM, respectively. No detectable binding of keratinocyte growth factor was observed. In studies aimed to determine whether FGF receptors contribute to the development of human tumors, we screened RNAs prepared from cell lines derived from a variety of solid tumors. High levels of the cl44 transcript were detected in 8 of 14 and 6 of 9 human mammary and kidney carcinomas, respectively, but only infrequently in other types of tumors. In contrast, FGFR1 was found to be frequently expressed in kidney, but not in breast tumor cells, suggesting a possible role for FGFR4 in human mammary cancer.

The lymphoproliferative disease virus of turkeys (LPDV) is the etiological agent of a rapidly developing lymphoproliferative process in turkeys. To better understand the genetic relationships of LPDV to other retroviruses we determined the nucleotide sequence of its pol gene. Comparative computer analyses of the deduced amino acid sequences of the reverse transcriptase and integrase domains within pol established that LPDV represents a distinct class of avian retroviruses that is most closely related to the avian leukemia-sarcoma viruses.

Virulent, wild-type equine infectious anemia virus (EIAV) is restricted in one or more early steps in replication in equine skin fibroblast cells compared with cell culture-adapted virus, which is fully competent for replication in this cell type. We compared the sequences of wild-type EIAV and a full-length infectious proviral clone of the cell culture-adapted EIAV and found that the genomes were relatively well conserved with the exception of the envelope gene region, which showed extensive sequence differences. We therefore constructed several wild-type and cell culture-adapted virus chimeras to examine the role of the envelope gene in replication in different cell types in vitro. Unlike wild-type virus, which is restricted by an early event(s) for replication in equine dermis cells, the wild-type outer envelope gene chimeras are replication competent in this cell type. We conclude that even though there are extensive sequence differences between wild-type and cell culture-adapted viruses in the surface envelope gene region, this domain is not a determinant of the differing in vitro cell tropisms.

Attempts to isolate full-length unintegrated circular forms of the caprine arthritis encephalitis virus (CAEV) genome yielded only a large number of molecules with deletions. The 3' borders of most of these deletions were near the U3 region of the long terminal repeat whereas the 5' edges were found at various upstream sites within pol or env. With one exception, gag sequences were always present. Analysis of molecular clones derived from integrated proviral CAEV genomes from the same infected cells showed a similar spectrum of deletions. The presence of transcriptionally active elements within the U3 domain of the defective genomes, as well as cis-acting elements within the leader sequences known to be required for efficient encapsidation of viral RNA, suggested that the defective viral DNA genomes could be transcribed into defective RNA molecules which could then be packaged into virions. Isopycnic density gradient centrifugation of supernatants of infected cell cultures indicated the presence of particles with densities less than that expected for intact virions (1.16 g/cc). Northern analysis revealed the presence of smaller viral-specific RNAs that lacked env sequences. These data, along with the structures of the molecular clones, suggest that CAEV stocks contain particles with defective genomes. The role of these particles in influencing the course of virus infection remains to be determined.

Nucleotide sequence analysis of a cDNA library of EIAV-infected canine cells established a complex pattern of gene expression, characterized by alternatively spliced polycistronic transcripts. The EIAV tat gene product was shown to be encoded by at least three species of mRNA which differed in their ability to trans-activate the EIAV LTR upon expression in canine cells. The most active cDNA was monocistronic, consisting of three exons. The most abundant cDNA in the library contained four exons and was identical to a polycistronic transcript previously described (Noiman et al., 1990b) which contains open frames for Tat, putative Rev, and truncated transmembrane proteins. Products consistent in size with those predicted for these last two proteins could be detected in in vitro translation experiments. The third Tat message, another four-exon form, also potentially encodes an amino terminally truncated transmembrane protein. In vitro mutagenesis experiments and analysis of subgenomic and partial cDNA clones confirmed and extended previous findings that S1 sequences are essential for trans-activation and that Tat translation initiates at a non-AUG codon either in the full-length Tat message or in the genomic S1 open reading frame. The Tat protein (8 kDa) was detected in cells transfected with a Tat cDNA construct and in canine cells persistently infected with EIAV. The Tat activity of polycistronic mRNAs was lower than that of the monocistronic form, suggesting that the expression of the EIAV trans-activator may be subject to several levels of posttranscriptional control.

The pattern of expression of the caprine arthritis encephalitis virus genome (CAEV) in acutely infected tahr lung cells was found to be complex and temporally regulated. Employing Northern analysis, five CAEV-specific transcripts, 9, 6.5, 5.0, 2.5, and 1.4 kb, were detected. Nucleotide sequence analysis established the genetic structure of two species of cDNA, isolated from a library of CAEV-infected tahr cells, and suggested that they represent rev-like transcripts. One of these cDNA species was composed of three exons--the leader, an exon derived from the 5' region of env, and an exon which spanned the 3' orf. The second cDNA species consisted of four exons--three of which were identical with those of the former species. The additional exon (the second) was located at the 3' end of pol. These transcripts could potentially encode three proteins--a Rev-like protein, which is a fusion of 38 amino acids derived from the N-terminus of env and 91 residues from the 3' orf; a truncated form of the env transmembrane protein, and a novel protein, designated X composed of 73 amino acids. Thus, CAEV, like other lentiviruses, displays a complex pattern of gene expression, characterized by alternative splicing and the production of potentially polycistronic transcripts.

Fibroblast growth factors (FGFs) are polypeptide mitogens that induce the proliferation of a wide variety of cell types. Of the seven family members, the best characterized are basic and acidic FGF. In addition to their mitogenic effects, they participate in angiogenesis, differentiation and maintenance of survival of neurons, cell migration and embryonal development. Of all family members, keratinocyte growth factor (KGF) is unique in that it is a specific mitogen for epithelial cells and does not interact with the FGF receptor of fibroblasts. To study the interactions between KGF and its receptor, we isolated KGF and FGF receptors from keratinocytes and fibroblasts, respectively. In the course of this study, we isolated five different variants of the FGF receptor from human fibroblasts and showed that all were derived from a single genetic locus. Four of these variants encode transmembrane receptors and can be divided into two subgroups that differ from one another with respect to the number (two or three) of immunoglobulin (Ig)-like domains. Within each subgroup, one receptor differed from the other by the presence of a two-codon insertion. Thus, all the variations among the four isoforms are localized to their ligand binding domains. The fifth isoform encodes a molecule truncated just 3' to the first Ig-like domain and thus could be secreted from the cell. The transcripts encoding the long and short isoforms were found to be expressed in many cell types, but their relative levels of expression varied greatly depending on the cell type. These findings indicate that alternative splicing generates diverse FGF receptor isoforms in human cells.

Proto-dbl is a human proto-oncogene, whose oncogenic activation was initially detected by DNA transfection. We report significant sequence similarity between the predicted proto-dbl product and the products of CDC24, a Saccharomyces cerevisiae cell division cycle gene required for correct budding and establishment of cell polarity, and bcr, a gene implicated in the pathogenesis of chronic myelogenous leukemia (CML). Of 925 residues of the predicted proto-dbl protein, a stretch of 238 residues showed 29% and 22% identity over a region of similar length of the CDC24 and bcr proteins, respectively. When evolutionarily conservative substitutions were taken into account, the similarities were 68.8% and 71.6% for proto-dbl/CDC24 and proto-dbl/bcr gene products, respectively. Moreover, all three sequences were predicted to be markedly hydrophilic over this region. Very small deletions within the conserved region completely abolished transforming activity of dbl, while extensive deletion outside of this region had no effect. Even substitutions over a small stretch of close similarity with the other proteins substantially impaired transforming activity. Cells transformed by the dbl oncogene, like cdc24 mutants arrested at the nonpermissive temperature, form multinucleate cells. Thus, our findings indicate that the conserved region is an essential domain that may reflect important functional similarities among these otherwise highly divergent molecules.

The lymphoproliferative disease virus (LPDV) is the etiological agent of a lymphoproliferative disease that naturally occurs in turkeys. Recently, we have cloned the LPDV provirus and established it as a replication-competent genome devoid of a viral oncogene [Gak et al., J. Virol. 63 (1989) 2877-2880]. This report presents the nucleotide sequence of its long terminal repeat (LTR) and establishes it as a potent transcriptional element. Several features of the LPDV LTR were similar to those found in the LTRs of the avian sarcoma-leukemia viruses (ASLV) and include the primer-binding site (tRNATrp), the polypurine tract, the organization of the polyadenylation signal, the complexities of the U3, R and U5 regions, as well as a potential secondary structure in U5-R. The LTR sequence diverges significantly from the ASLV LTRs, which share a common structure and have extensive sequence homology mainly in the R and U5 domains. These findings support the conclusion that LPDV represents a distinct class of avian retrovirus, evolutionarily related to the ASLV family.

Equine infectious anemia virus (EIAV), a lentivirus, encodes a trans-activator (tat) which stimulates gene expression directed by the viral long terminal repeat (LTR). This function has been previously shown by us and others to be encoded by sequences within the middle region of the EIAV genome in which two short open reading frames, S1 and S2, reside. In the present study, by using in vitro mutagenesis, we show that disruption of S1, but not S2, completely abolished trans-activation. Addition of oligonucleotides complementary to S1 to cells transfected with a tat expression vector resulted in inhibition of trans-activation. EIAV cDNAs were isolated from a library of EIAV-infected cells constructed by using a eukaryotic expression vector. One cDNA clone which contained S1 sequences was able to trans-activate the EIAV LTR. Sequence analysis of this cDNA clone revealed that, in addition to S1, two other open reading frames were present. The cDNA still retained its activity when the latter two sequences were deleted.

The lymphoproliferative disease virus (LPDV) of turkeys is the retroviral agent of etiology of a rapidly developing, naturally occurring, lymphoproliferative process. Recently we have molecularly cloned the viral genome. The lack of a susceptible cell culture which can sustain LPDV replication hampered the analysis of the infectious capability of the cloned genome. Based on the efficient in-vivo replication of LPDV we have developed a sensitive in-vivo approach aimed at establishing the infectious capability of the cloned provirus. According to this approach, peripheral leukocytes withdrawn from 3-week-old turkeys were transfected with the cloned DNA and the transfected leukocytes were re-injected into the turkey from which they had been obtained. The injected leukocytes enabled the efficient expression of the viral genome and the release into the blood stream of LPDV virions, which thereafter could travel to their appropriate in-vivo target lymphoid cells and start multiple replication cycles, resulting in the development of a detectable viremia. The applicability of this in-vivo assay for other cloned viral genomes is discussed.

The pattern of expression of the equine infectious anemia virus (EIAV) genome in a persistently infected canine cell line was determined. Five EIAV-specific transcripts (8.2, 5.0, 4.0, 2, and 1.8 kilobases [kb]) were detected by using subgenomic restriction enzyme fragments of EIAV DNA and EIAV-specific oligonucleotides as probes. The 8.2-kb mRNA could be shown to represent viral genomic RNA, whereas the smaller transcripts were generated by splicing events. Evidence was obtained that indicated that each subgenomic RNA species shared a common 5'-splice donor. The 5.0-kb mRNA was found to be expressed at relatively low levels, was difficult to detect consistently, and appeared to be generated by a single splicing event which linked the 5' exon to the 3' region of pol. The 4.0-kb transcript was concluded to be the env mRNA on the basis of its hybridization pattern with the various probes and its abundance. The 2-kb species was found to be multiply spliced and was encoded by sequences derived from orf2 but was not detected by probes representing 3'-env/3'-orf sequences. The 1.8-kb species was shown to consist of sequences representing orf1, part of orf2, and the 3'-orf/env and may represent the message for the EIAV trans-activator gene.

Cells of the established MvlLu mink line spontaneously released a reverse transcriptase-containing virus after long-term passage in tissue culture. By molecular hybridization, DNA of normal mink cells was found to possess extensive nucleotide sequence homology with a reverse-transcription product of the viral genome, demonstrating that the new isolate was an endogenous virus of mink origin. The mink virus shared antigenic determinants with the major structural proteins of known mammalian type C viruses. Double-antibody competition radioimmunoassays were developed by utilizing the purified major structural protein, p30, of the mink endogenous virus. The virus was shown to possess antigenic determinants unique from those of other known mammalian type C viruses. It exhibited a higher degree of immunological cross-reactivity with endogenous rat type C and horizontally transmitted feline leukemia viruses than with other mammalian type C viruses tested. The finding that mink cells can remain nonvirus producing for many cell generations argues that there normally exists some cellular restriction to endogenous virus expression in this species.

Type C RNA viruses have been isolated from several Old World vertebrates, and an even larger number of Old World species have been shown to contain endogenous viral genetic sequences. The present report describes the first isolation of type C virus endogenous to a species originating in the New World. This virus, isolated from cells of the Columbian black-tailed deer, Odocoileus hemionus, is shown to possess genetic sequences in common with DNA of its species of origin. While it shares biochemical and immunologic characteristics with other mammalian type C viruses, its immunologic properties readily distinguish it from known endogenous viruses.

Clonal isolates of an early passage stock of woolly monkey sarcoma virus (WSV) have been shown to code for different numbers of woolly monkey helper leukemia virus gag gene products. In the present report, the molecular mechanisms responsible for their differential expression of gag gene products have been analyzed. Three WSV RNA genomes were shown to possess sedimentation coefficients consistent with the differences demonstrated in their allotments of helper viral sequences. The WSV variant (WSV clone 9) that expressed no detectable proteins was shown to contain the largest amount of helper viral information. Moreover, there was no additive hybridization of the WLV complementary DNA probe by RNA of this WSV clone and that of a WSV clone coding for several gag gene products. These results suggest that the lack of expression of gag gene products by WSV clone 9 is not due to a major deletion of helper viral gag gene sequences. Similar levels of WLV-specific RNA were demonstrated in cells nonproductively transformed by each WSV clone, arguing that the ability to express gag gene proteins was not related to the magnitude of viral RNA transcription. Taken together, the results are most consistent with a mechanism by which small deletions or point mutations in the genomes of some WSV variants result in premature termination of translation or synthesis of immunologically nonreactive gag gene proteins. The present findings have implications concerning the effects of evolutionary selective pressures on helper viral genetic information in mammalian transforming viruses.

We determined the nucleotide sequence of the v-H-ras-related oncogene of BALB/c murine sarcoma virus. This oncogene contains an open reading frame of 189 amino acids that initiates and terminates entirely within the mouse cell-derived ras sequence. The protein encoded by this open reading frame matches the sequence predicted for the T24 human bladder carcinoma oncogene product, p21, in all but two positions. The presence of a lysine residue in position 12 of BALB/c murine sarcoma virus p21 likely accounts for its oncogenic properties.

Several temperature-sensitive mutants of the Rauscher strain of murine leukemia virus representing three distinct physiological groups have been further characterized. Genetic recombination between different pairs of these mutants has been demonstrated. Several representative genetic recombinants were isolated and shown to replicate equally well at the permissive (31 C) and nonpermissive (38 C) temperatures and to have serological characteristics of the wild-type parental virus. Alternative models for the mechanisms involved in recombination between type C RNA viruses are discussed.

A retrovirus previously isolated from a tumored Russell's viper is shown by molecular hybridization to be an endogenous virus of this reptilian species. Radio-immunologic techniques revealed that the viper retrovirus is immunologically and, hence, evolutionarily related to endogenous type D retorviruses of Old World primates. These findings extend the number of vertebrate classes possessing endogenous retroviruses and suggest that type D retroviruses may even be more widely distributed in nature than type C retroviruses.

Human DNA contains multiple copies of a novel class of endogenous retroviral genomes. Analysis of a human recombinant DNA clone (HLM-2) containing one such proviral genome revealed that it is a mosaic of retroviral-related sequences with the organization and length of known endogenous retroviral genomes. The HLM-2 long terminal repeat hybridized with the long terminal repeat of the squirrel monkey virus, a type D retrovirus. The HLM-2 gag and pol genes share extensive nucleotide sequence homology with those of the M432 retrovirus (a type A-related retrovirus), mouse mammary tumor virus (a type B retrovirus), and the avian Rous sarcoma virus (a type C retrovirus). Nucleotide sequence analysis revealed regions in the HLM-2 pol gene that were as much as 70 percent identical to the mouse mammary tumor virus pol gene. A portion of the putative HLM-2 env gene hybridized with the corresponding region of the M432 viral genome.

Murine Rauscher leukemia virus (MuRLV) from BALB/c plasma consisting of a mixture of an ecotropic and a xenotropic virus could be separated out by a selection process when propagated in human and simian cell cultures. This hypothesis is supported by obtaining consistently lower infectivity titers of human cell propagated RLV in human and simian cells as compared to MuRLV propagated in mouse cell cultures. Furthermore, RLV passaged in a simian cell culture failed to replicate in mouse cells, had a wide host range, was able to rescue Moloney sarcoma genome, possessed murine type C group-specific antigen, and was neutralized by anti-HRLV. Its reverse transcriptase was strongly inhibited by antiserum to MuRLV enzyme; however, antiserum to woolly monkey enzyme also inhibited (30%) its reverse transcriptase, suggesting some difference in antigenic properties. Inoculation of this virus in rhesus monkeys was inconclusive.

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.

A transforming gene isolated from T24 human bladder carcinoma cells is closely related to the BALB murine sarcoma virus (MSV) onc gene (v-bas). This transforming gene is localized to a 4.6 kilobase pair (kbp) region and is expressed as a 1.2-kbp polyadenylated transcript, which contains v-bas related sequences. Moreover, antisera known to detect the immunologically related onc gene products of BALB- and Harvey-MSVs recognized elevated levels of a related protein in T24 cells. The normal human homologue of v-bas was found to be indistinguishable from the T24 oncogene by heteroduplex and restriction enzyme analysis. These results imply that rather subtle genetic alterations have led to the activation of the normal human homologue of v-bas as a human transforming gene.

Molecular clones of the integrated form of the genome of equine infectious anemia virus (EIAV), the etiologic agent of a naturally occurring, worldwide disease of horses, were obtained. The restriction map of a full-length genome was determined. Additional evidence for the close evolutionary relationship between EIAV and a prototype lentivirus (caprine arthritis encephalitis virus) was acquired by Southern blotting and immunological analyses. An interspecies radioimmunoassay was developed in which EIAV and ovine and caprine lentiviruses could be detected equally well. These studies make available precisely defined reagents to pursue the study of the mechanisms of pathogenesis of lentiviral induced diseases.

The nucleotide sequence of the integrated form of the genome of the equine infectious anemia virus was determined. By comparison with LTR sequences of other retroviruses, signals for the control of viral gene transcription and translation could be identified in the EIAV LTR. Open reading frames for gag and pol genes were identified and their sequences matched very closely to those determined previously by others. However, in the present study, the pol gene reading frame was open throughout its entire length. The open reading frame for the env gene product was constructed from the sequences of two independent EIAV clones. Thus, a noninfectious genomic-length clone was shown to contain a frameshift mutation approximately in the middle of the presumed env gene coding sequence, whereas the sequence of another clone was open in this region. The deduced amino acid sequences of the EIAV gag and pol products showed closer evolutionary relationships to those of known lentiviruses than to other retroviruses. There was also partial sequence homology between predicted env gene products of EIAV, visna virus, and HTLV-III/LAV. Sequences analogous to the sor region of other lentiviruses could not be identified in our EIAV clone. A short open reading frame at the 3' end of the genome that overlapped env but not the 3' LTR was present but lacked significant sequence similarity to the 3' open reading frames of other lentiviruses. Thus, the sequence and general structure of EIAV most closely resemble those of known lentiviruses.

Type C RNA viruses have been isolated from a large number of mammalian species. These agents may be horizontally transmitted as infectious cancer-inducing agents, or vertically transmitted from one generation to the next, often in an unexpressed form, within the host genome. To date, the translational products of three viral genes have been identified. With purified virus-coded proteins as probes, sensitive and highly specific radioimmunologic assays have been developed for the detection of antibodies and antigens related to the known type C viruses. These techniques have proved valuable in sero-epidemiologic studies of the horizontally transmitted oncogenic viruses of cats, cattle, and gibbons, and have been used to detect translational products of endogenous viruses in tissues of species from which complete virus has yet to be isolated. This review describes the application of radioimmunoassays in the search for immunologic evidence of type C virus expression in man.

The cell-derived domain of Gardner-Rasheed feline sarcoma virus (GR-FeSV) consists of a gamma-actin- and a tyrosine-specific protein kinase-encoding sequence designated v-fgr. By utilizing a v-fgr probe, it was possible to detect related sequences present at low copy number in DNAs of a variety of mammalian species and to isolate a human fgr homologue. Comparative studies revealed that this human DNA clone represented all but 200 base pairs of v-fgr. Analysis of human genomic DNA demonstrated that the fgr protooncogene was distinct from the cellular homologues of other retrovirus onc genes. In addition, the fgr protooncogene was localized to the distal portion of the short arm of human chromosome 1 at p36.1-36.2 by in situ hybridization. Taken together, our findings establish that the fgr protooncogene is a unique member of the tyrosine kinase gene family.

The DBL oncogene was generated by rearrangements involving three discontinuous regions of the human genome. Analyses of panels of human X rodent somatic cell hybrids demonstrated that the DBL proto-oncogene located on the X chromosome (just proximal or distal to bands q26-27.2) underwent recombination at its 5' and 3' ends with sequences derived from chromosomes 3 (p13q-ter) and 16 (p13-q22), respectively. DBL was localized to chromosome Xq27-q28 by in situ hybridization. Another oncogene, MCF2, was previously shown to contain sequences derived from Xq27 as well. Comparison of the restriction maps and nucleotide sequences of genomic and cDNA clones representing the chromosome X-specific sequences of the DBL oncogene and MCF2, taken together with their chromosomal localization, indicates that these oncogenes were derived from the same genetic locus.

A reverse transcriptase-containing virus has recently been isolated from a squirrel monkey (Saimiri sciureus). Molecular hybridization studies demonstrate that the squirrel monkey retrovirus (SMRV) is endogenous to this New World primate, yet lacks detectable nucleotide sequence homology with cellular DNAs of representative Old World primates or with the genomes of previously isolated Old World primate retroviruses. The 35,000-dalton major structural protein (p35) of SMRV was purified and shown to possess antigenic determinants distinct from those of known retroviruses. While SMRV was found to lack antigenic determinants broadly shared among mammalian type-C viruses, immunologic crossreactivity was demonstrated between SMRV p35 and the major structural protein (p26) of Mason-Pfizer monkey virus, a prototype type-D retrovirus of Old World monkeys. These findings support the concept that SMRV and Mason-Pfizer monkey virus are evolutionarily related, and raise the possibility that a progenitor of type-D retroviruses became genetically associated with primates at a very early time in their evolution.

An N-ras-related transforming gene was detected in the human lung carcinoma cell line SW-1271 and molecularly cloned. The lesion responsible for its acquisition of transforming activity was localized to a single nucleotide transition from A to G in codon 61 of the predicted protein. This lesion in the second exon results in the substitution of arginine for glutamine at this position. These findings, together with previous studies, indicate that the activation of ras oncogenes in human tumors is most commonly due to point mutations at one of two major "hot spots" in the ras coding sequence.

An N-ras transforming gene was detected in human rectal carcinoma-derived cells (7060) and molecularly cloned. The genetic lesion responsible for the transforming activity of the 7060 oncogene was localized to a single nucleotide transition from A to T in codon 61 of the predicted protein. This lesion in the second exon results in substitution of histidine for glutamine at this position. We also found an EcoRI restriction fragment length polymorphism, consisting of two alleles, of the human c-myb gene. The 7060-transformed epithelial cells showed the homozygous phenotype, while normal fibroblasts of the same patient showed the heterozygous phenotype. This suggests a relationship between the phenotypic change in the c-myb locus and the induction of the 7060 tumor.