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Down-regulated by mos (Drm)/Gremlin is a highly conserved protein whose properties and expression pattern suggest a role in early development, tissue-specific differentiation, and cell transformation. We have investigated the biosynthesis and processing of Drm expressed endogenously in rat fibroblasts or overexpressed following transient or stable transfection. Analysis of metabolically labeled cells revealed that Drm exists in secreted and cell-associated forms that exhibit similar mobilities in SDS-polyacrylamide gel electrophoresis. Protein analysis indicated that Drm is present in two major species: a slow migrating glycosylated form and a nonglycosylated form. Both forms of Drm are able to undergo phosphorylation. Drm is released into the media within 30 min of synthesis and is detectable for up to 4-5 h, whereas the cell-associated form has a half-life of about 1 h. Confocal immunofluorescent microscopy indicates that Drm is present both on the external surface of expressing cells, as well as within the endoplasmic reticulum and the Golgi. Both glycosylated and nonglycosylated forms of Drm exhibit identical distributions and are able to antagonize bone morphogenetic protein signaling. Like the soluble form, the cell-associated forms are capable of binding (125)I-bone morphogenetic protein-4. These properties are consistent with a role for Drm in interfering with signaling and indicate that Drm may act at the cell surface during tissue development and transformation.

Non‐dividing neuroretina cells from chicken embryos are induced to proliferate after a long latency, following infection with Rous associated virus type 1, an avian retrovirus which does not carry a transforming gene. We have isolated from these proliferating cells an acutely mitogenic retrovirus, designated IC10, which contains a novel oncogene. Nucleotide sequencing showed that the IC10 virus has transduced 1101 nucleotides of cellular origin inserted between the gag and env genes of RAV‐1. This oncogene, designated v‐Rmil, is 70.1% homologous to v‐mil. v‐Rmil encodes a protein of 40,976 daltons sharing 83.8% homology with the catalytic domain of the v‐mil protein. Divergence with the v‐mil gene product is observed at the NH2‐ and COOH‐terminal portions of the v‐Rmil protein. Restriction analysis of normal chicken DNA indicated that v‐Rmil is derived from a cellular gene distinct from c‐mil. The c‐Rmil gene is transcribed through a major mRNA, greater than 10 kb in length, that is detected at much higher levels in neuroretinas, as compared to other embryonic tissues.

Rous sarcoma virus (RSV) stimulates in quail embryo neuro‐retina (NR) cultures the specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma‐aminobutyric acid, a major inhibitory neurotransmitter in NR and in central nervous system. In quail embryo NR cultures transformed by ts NY‐68, a thermodependent transformation‐defective mutant of RSV, stimulation of GAD activity is regulated by pp60v‐src, the product of the src gene of RSV. Fibroblasts and myoblasts have a very low GAD activity that is not stimulated after transformation by RSV. Neuronal clones, previously derived from ts NY‐68‐transformed established NR cell lines, have a high GAD activity which is regulated by pp60v‐src, while other clones have a low GAD activity apparently not regulated by pp60v‐src. These data indicate that pp60v‐src selectively activates the expression of GAD in distinct neuronal cells of quail embryo NR cultures transformed by RSV. GAD activity is also stimulated in NR cells infected with viruses containing v‐mil.

c-Rmilis the cellular allele of the v-Rmiloncogene, transduced during in vitropassaging of Rous associated virus type 1 in chicken embryonic neuroretina (NR) cells. The c-Rmilproto-oncogene is the avian homolog of the mammalian B-rafgene and belongs to the mil/rafoncogene family of serine/threonine protein kinases. We recently reported that the avian c-Rmilgene encodes two proteins of 94 and 95 kDa, resulting from an alternative splicing mechanism. We describe here the exon-intron organization of the coding region of the chicken c-Rmillocus. We show that c-Rmilproteins are encoded by 19 exons lying within about 100 kbp of genomic DNA. Comparison of the organization of this gene with those of the other mil/rafgenes shows strong similarities within three conserved domains previously identified in mil/rafprotein kinases. However, c-Rmilcontains two additional 5′ coding exons that are not present in the other mil/rafgenes.

The lens-specific proteins alpha and delta crystallins and lentoid bodies, structures that follow a differentiation pathway similar to that of the lens, regularly appear after 4 to 5 weeks in quail embryo neuroretina monolayer cultures. We have investigated the effects of the avian oncogenic retroviruses Mill Hill 2 and Rous sarcoma virus on this process. Quail embryo neuroretina cells transformed by Mill Hill 2 virus were established into permanent cultures that synthesized alpha and delta crystallins and contained stem cells for the production of lentoid bodies. In contrast, transformation with the Rous sarcoma virus mutant tsNY-68 blocked the appearance of mRNA crystallins, but cytoplasmic alpha and delta crystallin mRNA and alpha crystallin appeared 44 h after a shift to the nonpermissive temperature. However, delta crystallins and lentoid bodies were only present after 7 days. The crystallins of transformed quail neuroretina cultures were immunologically indistinguishable from those of quail lenses and of normal quail embryo neuroretina cultures.

We previously reported that infection of chicken embryonic neuroretina cells with Rous-associated virus type 1 leads to the frequent occurrence of spliced readthrough transcripts containing viral and cellular sequences. Generation of such chimeric transcripts constitutes a very early step in oncogene transduction. We report, here, the isolation of a c-mil transducing retrovirus, designated IC4, which contains a highly mutated U3 sequence in which 48% of A is converted to G. Functional analysis of this variant U3 indicated that these mutations do not impair viral transcription and replication; however, they abolish functioning of its polyadenylation signal, thus allowing readthrough transcription of downstream cellular sequences. On the basis of these results, we designed a nonreplicative retroviral vector, pIC4Neo, expressing the neomycin resistance (Neo(r)) gene under the control of the IC4 long terminal repeat. Infection of nondividing neuroretina cells with virus produced by a packaging cell line transfected with pIC4Neo occasionally resulted in sustained cell proliferation. Two independent G418-resistant proliferating cultures were found to express hybrid RNAs containing viral and cellular sequences. These sequences were characterized by reverse transcription-PCR and were identified in both cultures, suggesting that proliferation was correlated with a common integration locus. These results indicate that IC4Neo virus functions as a useful insertional mutagen and may allow identification of genes potentially involved in regulation of cell division.

Non‐dividing neuroretina cells from chicken embryos are induced to proliferate after a long latency, following infection with Rous associated virus type 1, an avian retrovirus which does not carry a transforming gene. We have isolated from these proliferating cells an acutely mitogenic retrovirus, designated IC10, which contains a novel oncogene. Nucleotide sequencing showed that the IC10 virus has transduced 1101 nucleotides of cellular origin inserted between the gag and env genes of RAV‐1. This oncogene, designated v‐Rmil, is 70.1% homologous to v‐mil. v‐Rmil encodes a protein of 40,976 daltons sharing 83.8% homology with the catalytic domain of the v‐mil protein. Divergence with the v‐mil gene product is observed at the NH2‐ and COOH‐terminal portions of the v‐Rmil protein. Restriction analysis of normal chicken DNA indicated that v‐Rmil is derived from a cellular gene distinct from c‐mil. The c‐Rmil gene is transcribed through a major mRNA, greater than 10 kb in length, that is detected at much higher levels in neuroretinas, as compared to other embryonic tissues.

We reported previously that post-mitotic chicken embryonic neuroretina (NR) cells are induced to proliferate following in vitro infection with RAV-1, a retrovirus that does not carry an oncogene. NR cell multiplication results from the frequent activation and subsequent retroviral transduction of two related serine/threonine protein kinases, the c-mil/c-raf or c-Rmil/B-raf genes. We also showed that a very early event in the activation of these proto-oncogenes is the synthesis of chimeric mRNAs containing viral and cellular sequences joined by a splicing mechanism. In the current study, we have examined the ability of RAV-1 to induce proliferation of quail NR cells. By using the reverse transcription-polymerase chain reaction technique, we identified, in several proliferating quail NR cultures infected with RAV-1, a chimeric mRNA containing cellular sequences joined to the RAV-1 splice donor site. These cellular sequences are derived from a gene designated R10, which is expressed through a 1.9-kilobase (kb) mRNA detected in several embryonic tissues. A second transcript of 2.3 kb is specifically expressed in the NR, where both transcripts are developmentally regulated. The R10 cDNA encodes a 251-amino acid polypeptide that contains a leucine zipper motif. It exhibits significant similarity with the putative D52/N8L protein, encoded by an mRNA reported previously to be overexpressed in human breast and lung carcinomas. By using polyclonal antibodies specific for its amino-terminal and leucine zipper-containing regions, we identified the R10 gene product as a cytoplasmic protein of 23 kDa in cultured avian fibroblasts. A second protein of 30 kDa is detected in post-mitotic NR cells that express the 2.3-kb transcript. We also show, by in vitro transcription/translation and immunoprecipitation, that the R10 protein can readily form homodimers, presumably through its leucine zipper motif.

The c-Rmil/B-raf proto-oncogene is a member of the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair alternatively spliced exon located between exons 8 and 9 and, therefore, designated exon 8b. Human and mouse B-raf mRNAs also contain the 120-base pair alternatively spliced exon 10 previously described in the avian c-Rmil gene. Independent splicing of these two exons, located between the conserved region 2 (CR2) and the catalytic domain (CR3) gives rise to mRNAs potentially encoding four distinct proteins. By using specific sera generated against different portions of B-Raf, we identified at least 10 protein isoforms in adult mouse tissues. Some isoforms, in the range of 69-72 kDa, are not recognized by antisera directed against peptides encoded by exons 1 and 2, indicating the existence of B-Raf proteins with two different NH2 extremities. The other isoforms, in the range of 79-99 kDa, contain the amino acids encoded by exons 1 and 2, by either or both of the alternatively spliced exons, and, possibly, by another of the unidentified exon. Analysis of B-raf mRNA expression by reverse transcriptase-polymerase chain reaction and immunocharacterization of B-Raf proteins in different tissues of the adult mouse showed a tissue-specific pattern of B-Raf isoforms expression. Interestingly, isoforms containing amino acids encoded by exon 10 are specifically expressed in neural tissues. Taken together, these results suggest that distinct B-Raf proteins could be involved, in a tissue-specific manner, in signal transduction pathways.

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP‐stimulated MAP kinase activation. We show that cAMP stimulates p44mapkindependently of these events. Secondly, we studied the role of B‐Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B‐Raf as well as an upward shift in its electrophoretic mobility. We show that B‐Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK‐1. However, cAMP inhibits B‐Raf autokinase activity as well as its ability to phosphorylate and activate MEK‐1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B‐Raf in vitro. Further, we show that B‐Raf binds to p21ras, but more important, this binding to p21rasis virtually abolished with B‐Raf from PC12 cells treated with CPT‐cAMP. Hence, these data indicate that the PKA‐mediated phosphorylation of B‐Raf hampers its interaction with p21ras, which is responsible for the PKA‐mediated decrease in B‐Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.

Neuroretinas from 6–7 day-old chick embryos were cultivated after trypsin dissociation as monolayer cultures in Petri dishes, and examined after various intervals of time with the electron microscope. Soon after plating, cells begin to reaggregate in small clumps, and typical rosettes are formed. During the first week in vitro, cells appear to differentiate as neuroblasts and presumed Müller cells; the latter form a continous sheet on the substrate, upon which neuroblasts migrate and grow their neurites. Differentiated ribbon synapses are found after 8 days in vitro, the time at which they normally appear in situ. After 15 and 21 days in vitro, synapses are still found in large numbers, mimicking their “in vivo” counterparts. Photoreceptor cells were identified on the basis of the presence of typical ribbons in their cytoplasm, but no outer segment was found. It appears then that synaptogenesis in the retina is programmed independently of the tissue environment, which is markedly disturbed in the monolayer culture.

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44 mapkindependently of these events. Secondly, we studied the role of B-Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B-Raf as well as an upward shift in its electrophoretic mobility. We show that B-Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK-1. However, cAMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21 ras, but more important, this binding to p21 rasis virtually abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21 ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.

Using differential display analysis, we compared the expression of RNA in v-mos-transformed cells and their flat revertant and isolated a novel gene, drm (down-regulated in mos-transformed cells), whose expression is down-regulated in parental v-mos-transformed cells but which is expressed at a high level in the revertant and normal rat fibroblasts (REF-1 cells). Analysis of different oncogene-transformed cells revealed that drm gene expression was also suppressed in REF-1 cells transformed by v-ras, v-src, v-raf, and v-fos. The drm cDNA contains a 184-amino-acid-protein-encoding open reading frame which shows no significant homologies to known genes in DNA databases. Polyclonal antibodies raised against drm peptide detect a protein with the predicted size of 20.7 kDa in normal cells and under nonpermissive conditions in cells conditionally transformed by v-mos but not in parental v-mos-transformed cells. Northern analysis of normal adult tissues shows that drm is expressed as a 4.4-kb message in a tissue-specific manner, with high expression in the brain, spleen, kidney, and testis and little or no expression in the heart, liver, and skeletal muscle. In situ hybridization analysis in adult rat tissue reveals good correlation with this pattern and indicates that drm mRNA is most highly expressed in nondividing and terminally differentiated cells, such as neurons, type 1 lung cells, and goblet cells. Transfection of a drug-selectable drm expression vector dramatically reduced the efficiency of colony formation in REF-1 and CHO cells, and the drm-transfected REF-1 survivors expressed low or nondetectable levels of exogenous drm mRNA. The toxic effects of drm can be overcome by cotransfection with constructs expressing oncogenic ras; furthermore, cells expressing high levels of drm and conditionally transformed with mos-expressing Moloney murine sarcoma virus rapidly undergo apoptosis when shifted to the nonpermissive temperature. Taken together, our data suggest that cells expressing high levels of drm undergo apoptotic death in the absence of oncogene-induced transformation and that drm represents a novel gene with potential roles in cell growth control or viability and tissue-specific differentiation.

Developmental control of gene expression often results from the coupling of growth arrest with the establishment of differentiation programs. QR1 is a gene specifically expressed in retinas during the late phase of embryogenesis. At this stage neuroectodermal precursors have reached terminal mitosis and are undergoing differentiation into distinct cell types. Transcription of the QR1 gene is tightly regulated during retinal development: this gene is expressed between embryonic day 9 (ED9) and ED17 and is completely repressed at hatching in quail. Moreover, QR1 transcription is downregulated when postmitotic neural retina cells are induced to proliferate by pp60v-src. We studied the stage-dependent transcriptional control of this gene during quail neural retina (QNR) cell development. Transient transfection experiments with QR1/CAT constructs at various stages of development showed that a region located between -935 and -1265 bp upstream of the transcription start site is necessary to promote transcription in retina cells during the late phase of embryonal development (QNR9, corresponding to ED9). By in vivo footprinting assays we identified at least two elements that are occupied by DNA-protein complexes in QNR cells: the A and B boxes. The A box allows formation of several biochemically distinct complexes: C1, C2, C3, and C4. Formation of the C2 complex mainly during early stages (ED7) and of C2, C3, and C4 complexes during postnatal life correlates with repression of QR1 transcription, whereas the C1 complex is strongly induced at ED11 when the QR1 gene is expressed. We previously showed that C1 was involved in downregulation of QR1 transcription by pp60v-src. Several complexes are also formed on the B box. We show that these complexes are exclusively present in neural tissues and that they involve members of the POU family of transcription factors. Mutations of each one of the two regions which abolish the binding of the C1 factor(s) on the A box and of the POU factor(s) on the B box also prevent stimulation of QR1 transcription in QNR9. Therefore, both elements appear to be required for the stage-specific transcription of the QR1 gene. We also show that the regulatory region from position -1265 to position -935 is able to confer stage-specific transcription upon a heterologous promoter (thymidine kinase). Indeed, this region stimulates transcription in differentiating retinas (QNR9) and represses transcription in terminally differentiated retinas (QNR17, corresponding to postnatal life). Our results suggest that cell growth regulation and developmental control are coordinated through the A and B boxes in regulating QR1 transcription during retinal differentiation.

We previously described the isolation of the IC10 retrovirus which transduced the v-Rmil oncogene, a new member of the mil/raf gene family. This virus was generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina (NR) cells and was selected for its ability to induce proliferation of these nondividing cells. IC10 was isolated after six passages of culture supernatants but was not detected in proliferating NR cells during early virus passages. In this study, we molecularly cloned and sequenced another v-Rmil-containing provirus, designated IC11, from NR cells infected at the third virus passage of the same experiment. Both IC11 and IC10 transduced only the serine/threonine kinase domain of c-Rmil. Comparison of v-Rmil and c-Rmil sequences indicated that amino-terminal truncation is sufficient to activate the mitogenic properties of c-Rmil. IC11 and IC10 have identical 3' ends but differ by their 5' RAV-1-Rmil junctions. The 3' ends of both viruses were generated by recombination between Rmil and env genes, involving partial sequence identity. The 5' RAV-1-Rmil junction of IC11 was formed by a splicing process between the RAV-1 leader and a 37-bp c-Rmil exon located upstream of the kinase domain. NR cells infected with this virus synthesize a unique Rmil protein. IC10 contains most of the gag gene recombined with v-Rmil and encodes a gag-Rmil hybrid protein. Serial passaging of IC11 in NR cells led to the formation of a gag-Rmil-containing retrovirus. These results indicate that IC11 represents an early step in transduction and that this virus further recombined with RAV-1 to generate IC10. They confirm our previously proposed model for the multistep generation of v-mil-transducing retroviruses. Therefore, activation and transduction of c-mil and c-Rmil, in NR cells infected with RAV-1, result from a common mechanism.

When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src.

Expression of the src gene of Rous sarcoma virus in chicken embryo neuroretinal cells results in morphological transformation and sustained proliferation of this normally resting cell population. PA101 and PA104 are two mutants of Rous sarcoma virus which induce neuroretinal cell proliferation in the absence of morphological transformation. Their mitogenic property is temperature sensitive, and they both encode p60src proteins with low kinase activity. To study the role of the mitogenic function and protein kinase activity of p60src in tumorigenesis, we investigated the oncogenicity of PA101 and PA104. Both mutants were less tumorigenic than wild-type virus when injected into chicks. Tumorigenicity was further assayed by inoculating infected chicken embryo fibroblasts and neuroretinal cells onto the chorioallantoid membrane of embryonated duck eggs. This system provides a nonpermissive and immunodeficient environment for xenogenic cell grafting and allows the study of cell tumorigenicity within a temperature range of 37 to 39.5 degrees C. Chicken embryo fibroblasts and neuroretinal cells infected with PA101 were as tumorigenic as wild type-infected cells at 37 degrees C, but tumor development was significantly reduced at 39.5 degrees C. In contrast, both cell types infected with PA104 displayed sharply reduced tumorigenicity. Cell cultures derived from PA101 tumors induced on the chorioallantoid membrane were similar to the corresponding cells maintained in vitro in terms of morphology, production of plasminogen activator, relative amounts of phosphotyrosine in total cellular proteins, and phosphorylation of 34,000-molecular-weight protein. These results indicate that the expression of the mitogenic function of src does not account per se for cell tumorigenicity and that tumor formation is compatible with low levels of p60src protein kinase activity.

Expression of the src gene of Rous sarcoma virus (RSV) in chicken embryo neuroretinal (NR) cells results in morphological transformation and sustained proliferation of a normally resting cell population. We have previously reported the isolation of mutants of RSV which retain full growth-promoting activity while displaying reduced transforming properties. Two such mutants, PA101 and PA104, were used to investigate whether the p60src-associated kinase activity is required for the mitogenic function of src. A comparison of the patterns of phosphorylation of wild-type and mutant p60src revealed that the phosphorylation of tyrosine residues of p60src of PA104 was markedly reduced, whereas the relative amount of phosphotyrosine in p60src of PA101 was comparable to that of the wild-type protein. In vitro kinase activity of p60src immunoprecipitated from NR cells infected with PA101 or PA104 as measured by phosphorylation of the heavy chains of specific immunoglobulin G molecules was 1/10 that of the wild-type molecule. Moreover, when NR cells infected with mutants temperature sensitive for mitogenic capacity were maintained at a temperature either permissive or restrictive for cell growth, quantitation of kinase activity indicated that proliferation of NR cells could not be linked to the absolute level of in vitro kinase activity of p60src. Transformation of NR cells by wild-type RSV resulted in a 10-fold increase in total cellular phosphotyrosine and in the phosphorylation of tyrosine residues of a 34K protein, a possible in vivo substrate for p60src. In contrast, phosphorylation of tyrosine residues of cellular targets was markedly reduced in NR cells infected with PA101 or PA104. These results indicate that the mitogenic capacity of RSV in NR cells does not require elevated levels of p60src kinase activity.

The avian neuroretina (NR) is part of the central nervous system and is composed of photoreceptors, neuronal cells, and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors that differentiate after terminal mitosis and become organized in cell strata. Genes that are specifically expressed at the various stages of retinal development are presently unknown. We have isolated a quail (Coturnix coturnix japonica) cDNA clone, named QR1, encoding a 676-amino acid protein whose carboxyl-terminal portion shows significant similarity to those of the extracellular glycoprotein osteonectin/SPARC/BM40 and of the recently described SC1 protein. The QR1 cDNA identifies a mRNA detected in NR but not in other embryonic tissues examined. The levels of this mRNA are markedly reduced when nondividing NR cells are induced to proliferate by the v-src oncogene. QR1 expression in NR is limited to the middle portion of the inner nuclear layer, a localization that essentially corresponds to that of Müller cells. Transcription of QR1 takes place only during the late phase of retinal development and is shut off sharply at hatching. Signals that regulate this unique pattern of expression appear to originate within the NR, since the QR1 mRNA is transcribed in cultured NR cells and is shut off also in vitro at a time coinciding with hatching.

The embryonic avian neuroretina (NR) is part of the central nervous system and is composed of various cell types: photoreceptors and neuronal and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors which differentiate after terminal mitosis and become organized in cell strata. Proliferation of differentiating NR cells can be induced by infection with Rous sarcoma virus (RSV) and requires the expression of a functional v-src gene. To understand the mechanisms involved in the regulation of neural cell growth and differentiation, we studied the transcriptional regulation of QR1, a gene specifically expressed in postmitotic NR cells. Transcription of this gene is detected primarily in Müller cells and is strongly downregulated by the v-src gene product. Moreover, QR1 expression takes place only during the late phase of retinal development and is shut off abruptly at hatching. We have isolated a promoter region(s) of the QR1 gene that confers v-src responsiveness. By transfection of QR1-CAT constructs into quail NR cells infected with the temperature-sensitive mutant of RSV, PA101, we have identified a v-src-responsive region located between -1208 and -1161 upstream of the transcription initiation site. This sequence is able to form two DNA-protein complexes, C1 and C2. Formation of complex C2 is specifically induced in cells expressing an active v-src product, while formation of C1 is detected mainly in nonproliferating quail NR cells upon pp60v-src inactivation. C1 is also a target for regulation during development. We have identified the DNA binding site for the C1 complex, a repeated GCTGAC sequence, and shown that mutations in this element abolish binding of this factor as well as transcription of the gene at the nonpermissive temperature. Neither formation of C1 nor that of C2 seems to involve factors known to be targeted in the pp60v-src cascade. Our data suggest that C1 could be a novel target for both developmental control and oncogene-induced cell growth regulation.

We previously reported that serial passaging of Rous-associated virus type 1 in nondividing chicken embryo neuroretina cells leads to reproducible generation of acutely mitogenic retroviruses that transduced the catalytic domain of c-mil/c-raf or c-Rmil/B-raf. On the basis of structural analysis of several retroviruses, we proposed that the early step of oncogene transduction is the constitution of alternatively spliced leader-delta onc-poly(A) transcripts. Here, we show that neuroretina cells do synthesize hybrid leader-delta mil and leader-delta Rmil RNAs and that these RNAs exhibit mitogenic properties and serve as templates for the generation of transducing retorviruses.

dlPA105 is a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion in the N-terminal portion of the v-src gene coding sequence. This virus was isolated on the basis of its ability to induce proliferation of quiescent quail neuroretina cells. The altered v-src gene encodes a phosphoprotein of 45,000 daltons which possesses tyrosine kinase activity. DNA sequencing of the mutant v-src gene has shown that deletion extends from amino acid 33 to 126 of wild-type p60v-src. We investigated the tumorigenic and transforming properties of this mutant virus. dlPA105 induced fibrosarcomas in quails with an incidence identical to that induced by wild-type virus. Quail neuroretina cells infected with the mutant virus were morphologically transformed and formed colonies in soft agar. In contrast, dlPA105 induced only limited morphological alterations in quail fibroblasts and was defective in promoting anchorage-independent growth of these cells. Synthesis and tyrosine kinase activity of the mutant p45v-src were similar in both cell types. These data indicate that the portion of the v-src protein deleted in p45v-src is dispensable for the mitogenic and tumorigenic properties of wild-type p60v-src, whereas it is required for in vitro transformation of fibroblasts. The ability of dlPA105 to induce different transformation phenotypes in quail fibroblasts and quail neuroretina cells is a property unique to this Rous sarcoma virus mutant and provides evidence for the existence of cell-type-specific response to v-src proteins.

Expression of the v-src gene of Rous sarcoma virus in avian embryo neuroretina cells results in transformation and sustained proliferation of these normally resting cells. Transformed neuroretina cells are also tumorigenic upon inoculation into immunodeficient hosts. We have previously described conditional mutants of Rous sarcoma virus encoding p60v-src proteins which induce proliferation of neuroretina cells in the absence of transformation and tumorigenicity. These results suggest that p60v-src is composed of functionally distinct domains which may interact with multiple cellular targets. In this study, we describe a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion of 278 base pairs in the 5' portion of the v-src gene but which has retained the ability to induce proliferation of quail neuroretina cells. The deleted v-src gene encodes a 45,000-molecular-weight phosphoprotein which contains both phosphoserine and phosphotyrosine, is myristylated, and possesses tyrosine kinase activity indistinguishable from that of wild-type p60v-src. Molecular cloning and sequence analysis of the mutant v-src gene have shown that this deletion extends from amino acid 33 to 126 of the wild-type p60v-src. Therefore, this portion of the v-src protein is dispensable for the mitogenic activity of Rous sarcoma virus in neuroretina cells.

The avian neural retina (NR) is derived from proliferating neuroectodermal precursors which differentiate after terminal mitosis and become organized in cell strata. Proliferation of postmitotic NR cells can be induced by infection with Rous sarcoma virus (RSV) and requires the expression of a functional v-Src protein. QR1 is a retina-specific gene expressed exclusively at the stage of growth arrest and differentiation during retinal development. In NR cells infected with tsPA101, an RSV mutant conditionally defective in pp60v-src mitogenic capacity, QR1 expression is downregulated in proliferating cells at 37 degrees C and is fully restored when the cells become quiescent as a result of pp60v-src inactivation at 41 degrees C. We were able to arrest proliferation of tsPA101-infected quail NR cells expressing an active v-Src protein by serum starvation at 37 degrees C. This allowed us to investigate the role of cell growth in regulating QR1 transcription. We report that QR1 transcription is stimulated in growth-arrested cells at 37 degrees C compared with that in proliferating cells maintained at the same temperature. Growth arrest-dependent stimulation of QR1 transcription requires the integrity of the A box, a previously characterized cis-acting element responsible for QR1 transcriptional stimulation upon v-Src inactivation and during retinal differentiation. We also show that formation of the C1 complex on the A box is increased upon growth arrest by serum starvation in the presence of an active v-Src oncoprotein. Thus, the C1 complex represents an important link between cell cycle and developmental control of QR1 gene transcription during NR differentiation and RSV infection. By using antibodies directed against different Maf proteins of the leucine zipper family and competition with Maf consensus site-containing oligonucleotides in a gel shift assay, we show that the C1 complex is likely to contain a Maf-related protein. We also show that a purified bacterially expressed v-Maf protein is able to bind the A box and that the level of a 43-kDa Maf-related protein is increased upon growth arrest in infected retinal cells. Moreover, ectopic expression of c-mafI, c-mafII, and mafB cDNAs in quiescent tsPA101-infected quail NR cells is able to stimulate transcription of a QR1 reporter gene through the A box. Therefore, QR1 appears to be the first target gene for a Maf-related protein(s) in the NR.

IC1, IC2, and IC3 are novel c-mil transducing retroviruses generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina cells. They were isolated by their ability to induce proliferation of these nondividing cells. IC2 and IC3 were generated during early passages of RAV-1 in neuroretina cells, whereas IC1 was isolated after six consecutive passages of virus supernatants. We sequenced the transduced genes and the mil-RAV-1 junctions of the three viruses. The 5' RAV-1-mil junction of IC2 and IC3 was formed by a splicing process between the RAV-1 leader sequence and exon 8 of the c-mil gene. The 5' end of IC1 resulted from homologous recombination between gag and mil sequences. Reconstitution experiments showed that serial passaging of IC2 in neuroretina cells also led to the formation of a gag-mil-containing retrovirus. Therefore, constitution of a U5-leader-delta c-mil-delta RAV-1-U3 virus represents early steps in c-mil transduction by RAV-1. This virus further recombined with RAV-1 to generate a gag-mil-containing virus. The three IC viruses transduced the serine/threonine kinase domain of the cellular gene. Hence, amino-terminal truncation is sufficient to activate the mitogenic property of c-mil. Comparison of the transforming properties of IC2 and IC1 showed that the transduced mil gene, expressed as a unique protein independent of gag sequences, was weakly transforming in avian cells. Acquisition of gag sequences by IC1 not only increased the rate of virus replication but also enhanced the transforming capacity of the virus.

We report that nondividing neuroretina cells from chicken embryos can be induced to proliferate following infection with Rous-associated virus type 1 (RAV-1), an avian lymphomatosis retrovirus lacking transforming genes. Multiplication of RAV-1-infected neuroretina cells is observed after a long latency period and takes place initially in a small number of cells. We also show that serial virus passaging onto fresh neuroretina cultures leads to the generation of novel mitogenic viruses containing the mil oncogene. DNA analysis indicated that RAV-1 is the only provirus detected in cells infected at virus passage 1, whereas neuroretina cells infected at subsequent virus passages harbor mil-containing proviruses. Three viruses, designated IC1, IC2, and IC3, were molecularly cloned. Restriction mapping indicated that in each virus, truncated c-mil sequences were inserted within different portions of the RAV-1 genome. In addition, IC1 and IC2 viruses have transduced novel sequences that belong to the 3' noncoding portion of the c-mil locus. All three viruses induce neuroretina cell multiplication and direct the synthesis of mil-specific proteins. Proliferation of neuroretina cells infected at passage 1 of RAV-1 was not associated with any detectable rearrangement of c-mil, when a v-mil probe was used. However, these cells expressed high levels of an aberrant 2.8-kilobase mRNA hybridizing to mil but not to a long terminal repeat probe. Therefore, transcriptional activation of a portion of c-mil could represent the initial events induced by RAV-1 infection and lead to retroviral transduction of activated c-mil sequences.

Avian retrovirus MH2 contains two oncogenes, v-mil and v-myc. We have previously shown that a spontaneous mutant of MH2 (PA200-MH2), expressing only the v-mil oncogene, is able to induce proliferation of quiescent neuroretina cells. In this study, we investigated the transforming and tumorigenic properties of v-mil. PA200 induced fibrosarcomas in about 60% of the injected chickens, whereas inoculation of MH2 resulted mainly in the appearance of kidney carcinomas. Analysis of several parameters of transformation showed that PA200, in contrast to MH2, induced only limited in vitro transformation of fibroblasts and neuroretina cells. These results suggest that v-myc is the major transforming and tumorigenic gene in MH2-infected cells. This low in vitro transforming capacity differentiates v-mil not only from other avian oncogenes, but also from the homologous murine v-raf gene.

Previous studies showed that the amino-terminal domain of Rous sarcoma virus p60v-src involved in myristylation and membrane association of the protein is required for morphological transformation and anchorage independence. Analysis of src delection mutants revealed that the amino-terminal one-third of p60v-src, including the membrane-binding domain, is not essential for induction of cell proliferation. These results demonstrated that, in contrast to the cellular target(s) involved in morphological transformation and anchorage independence, the target(s) involved in mitogenic activity is accessible to nonmyristylated src proteins.

The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild-type src sequences. Three of the mutants, which were derived from the Schmidt-Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant.

The expression of p60v-src in chicken cells infected with Rous sarcoma virus causes stimulation of cell proliferation, morphological alteration, and anchorage independence. PA101 and PA104 are temperature-sensitive variants encoding mutant p60v-src proteins that are partially defective in the induction of these transformation parameters. To define the structural basis for the transformation defectiveness of the p60v-src mutants, the v-src genes of PA101 and PA104 were molecularly cloned and analyzed. Amino- and carboxy-terminal coding regions of the cloned mutant genes were exchanged with the corresponding regions of cloned wild-type v-src and chicken c-src genes, reconstructed into viral DNA, and expressed in infected cells maintained at various temperatures. This analysis revealed that lesions within the tyrosine kinase domains of the two mutant proteins confer temperature sensitivity on all three transformation functions of p60v-src. An amino-terminal region of the PA101 mutant protein, which coincides with the proposed modulatory domain and appears to interact with the kinase domain, affects morphological alteration in a temperature-independent manner. Our results suggest that the function of the kinase domain is essential to all three parameters examined, whereas the amino-terminal domain is important in determining cell morphology.

Rous sarcoma virus (RSV) stimulates in quail embryo neuro‐retina (NR) cultures the specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma‐aminobutyric acid, a major inhibitory neurotransmitter in NR and in central nervous system. In quail embryo NR cultures transformed by ts NY‐68, a thermodependent transformation‐defective mutant of RSV, stimulation of GAD activity is regulated by pp60v‐src, the product of the src gene of RSV. Fibroblasts and myoblasts have a very low GAD activity that is not stimulated after transformation by RSV. Neuronal clones, previously derived from ts NY‐68‐transformed established NR cell lines, have a high GAD activity which is regulated by pp60v‐src, while other clones have a low GAD activity apparently not regulated by pp60v‐src. These data indicate that pp60v‐src selectively activates the expression of GAD in distinct neuronal cells of quail embryo NR cultures transformed by RSV. GAD activity is also stimulated in NR cells infected with viruses containing v‐mil.

The genome of the avian retro virus MH2 contains, in addition to the v-myconcogene shared with three other avian retroviruses (MC29, CMII and OK-10), a second cell-derived oncogene, v-mil(refs 1–3). Like the three other viruses, which contain only v-myc, MH2 induces mainly liver and kidney carcinomas in fowl and transforms fibroblasts and macrophages in vitro4. However, MH2 and MC29 differ in their biological properties when assayed on cultures of chicken embryo neuroretina (NR) cells. Indeed, NR cells, which normally do not multiply in vitro, are induced to proliferate and become transformed upon infection with MH2, whereas infection with MC29 has no apparent effect on these cells5,6. To analyse the functions of the two oncogenes of MH2, we isolated spontaneous and in vitro-constructed mutants of this virus and investigated their effects on NR cell multiplication and transformation. We report here that expression of v-milis sufficient to induce NR cell proliferation, although it does not result in cell transformation. In addition, viruses expressing only the v-myconcogene fail to induce any detectable change in NR cells. However, cooperation of the two oncogenes is required to achieve transformation of NR cells by MH2.

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.

The avian neuroretina (NR) is composed of photoreceptors and different neurons that are derived from proliferating precursor cells. Neuronal differentiation takes place after terminal mitosis. We have previously shown that differentiating NR cells can be induced to proliferate by infection with Rous sarcoma virus (RSV) and that cell multiplication requires expression of a functional v-src gene. We speculated that the quiescence of NR cells could be determined by specific genes. Cell proliferation could then result from the negative regulation of these genes by the v-src protein. By differential hybridization of a cDNA library, we isolated eight clones corresponding to genes expressed in postmitotic NR cells from 13-day-old quail embryos, transcriptional levels of which are significantly reduced in NR cells induced to proliferate by tsNY68, an RSV mutant with temperature-sensitive mitogenic activity. Partial sequencing analysis indicated that one RNA encoded the calmodulin gene, whereas the other seven showed no similarity to known sequences. By using v-src mutants that induce NR cell proliferation in the absence of transformation, we showed that transcription of six genes was negatively regulated by the v-src protein and that of four genes was correlated with NR cell quiescence. We also report that a subset of genes are specifically transcribed in neural cells and developmentally regulated in the NR. These results indicate that the v-src protein regulates expression of genes likely to play a role in the control of neural cell growth or differentiation.

Rescue of SV40 virus after Sendai virus-mediated fusion of transformed mouse or hamster cell lines with permissive monkey cells was strikingly dependent on pH in the range 6.4-8.8, with a maximum at pH 8.4. The titer of virus recovered at pH 8.4 was 2 logs higher than that at pH 7.6, and 4 logs higher than that at pH 6.4. The pH-sensitive step was neither the number of heterokaryocytes formed, which was essentially the same at pH 7.6 and 8.4, nor the degree of SV40 replication in the monkey cells, which was also unaffected by pH variation in the range 7.2-8.4.