Increased sarcoma virus RNA in cells transformed by leukemia viruses: model for leukemogenesis.

A morphologically flat revertant of mink cells nonproductively infected with Moloney sarcoma virus exhibited contact inhibition and lacked detectable sarcoma virus RNA. Superinfection by usually nontransforming type C mammalian leukemia-causing viruses induced transformation and increased sarcoma virus RNA. The results suggest a model for leukemogenesis in animals by increasing, during replication of usually nontransforming leukemia viruses, the levels of RNA from potentially oncogenic cell or integrated virus transforming genes.

Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization.

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.

Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.

Multiple polyadenylation sites downstream from the human aFGF gene encoding acidic fibroblast growth factor.

We previously reported the isolation of two partial cDNA clones encoding human acidic fibroblast growth factor (aFGF). The nucleotide (nt) sequence throughout the coding region and the deduced amino acid sequence were presented [Jaye et al., Science 233 (1986) 541-545]. In this report, the isolation of additional aFGF cDNA clones and their nt sequence are presented. The human aFGF gene is shown to encode at least four functional polyadenylation sites and multiple regulatory sequences within the 3'-untranslated region. The aFGF open reading frame resides approx. 3100 bp upstream from the most frequently utilized 3' processing and polyadenylation site. Several less abundant cDNA clones provide evidence of polyadenylation at three less distal sites, which are colinear with genomic DNA. Northern-blot analysis reveals three detectable mRNA species, whose sizes and intensities correlate with the length and relative abundance of cDNA clones representing them.

Identification of novel peptide antagonists for von Willebrand factor binding to the platelet glycoprotein Ib receptor from a phage epitope library.

We have constructed a fusion phage epitope library in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different epitope bearing phage, has been used in an attempt to identify inhibitors of the von Willebrand factor (vWF)-platelet Glycoprotein Ib interaction. The library was screened with a monoclonal antibody (RG46) that recognizes the GPIb binding domain of vWF (amino acids 445-733). A total of 30 clones falling into 8 classes have been identified that react with the RG46 antibody. Isolates from all 8 classes are positive by immunoblot analysis. The amino acid sequence of the gene III fusion protein from positive clones showed a strong homology to the known RG46 epitope. Peptides identified from the screen were synthesized and used to demonstrate that some of the synthetic peptides exhibited inhibitory activity towards ristocetin induced binding of vWF to the GPIb receptor. Thus, we have demonstrated that screening a fusion phage epitope library with a monoclonal antibody that inhibits vWF binding to the GPIb receptor can be a useful tool not only for mapping antibody recognizing determinants, but also can serve as a source for identifying novel peptides that are antagonists for vWF binding to the platelet GPIb receptor.

Immunological relationship between the structural proteins of intracisternal A-particles of Mus musculus and the M432 retrovirus of Mus cervicolor.

A specific immunological relationship has been demonstrated between the main structural protein (p73) of intracisternal A-particles and a major internal protein (p24) of the M432 retrovirus.

Deletion mapping of moloney type C virus: polypeptide and nucleic acid expression in different transforming virus isolates.

The viral polypeptides and viral RNA present in cells transformed by various replication-defective type C viruses derived from Maloney murine leukemia virus were examined. Different portions of the Maloney type C viral genome were retained in the different transforming viruses, thus providing an opportunity for deletion mapping of the Moloney type C genome. DNA transcripts were prepared that are complementary to three distinct nonoverlapping portions of the Moloney viral geonome. Based on an anlysis of the polypeptides produced in the different transformed cells, one complementary DNA apparently respresents sequences coding for Moloney gp70; one complementary DNA represents a region of the Moloney genome common to all of the transforming viruses examined, and one complementary DNA represents the sequences for p30, p15, p10,12. A partial map of the different replication-defective transforming viruses is suggested.

Distribution of murine type B and type C viral nucleic acid sequences in template active and template inactive chromatin.

Template active chromatin and template inactive chromatin have been fractionated from mouse cells infected with the Moloney strain of murine leukemia virus. In vivo the cells produce abundant Rna homologous to Moloney leukemia virus, but do not produce either globin mRNA or RNA homologous to type B mouse mammary tumor virus. The DNA extracted from the template active chromatin or template inactive chromatin contained equal amounts of sequences homologous to Moloney type C virus, to type B virus, or to globin mRNA. The results are discussed with regard to the in vivo structure of chromatin and the difficulties in fractionating chromatin in vitro.

Immunochemical characterization of two major polypeptides from murine mammary tumor virus.

A major murine mammary tumor viral (MMTV) antigen, sl, originally described by Nowinski et al. (1967, 1968, 1971), has been purified from RIII mouse milk MMTV by sequential ion-exchange and gel chromatography. The purified protein with sl antigenic reactivity contains carbohydrate, and has an apparent minimal molecular weight of 52,000. It can be designated as gp52 (sl). Another major MMTV viral protein with a molecular weight of 27,000 has also been isolated, and antisera have been prepared against it. Both MMTV gp52 (sl) and p27 viral polypeptides have been iodinated with (125)I and used in immunoprecipitation and competition assays. The two MMTV proteins differ absolutely from each other and from major mouse type C viral polypeptides in molecular weight, immunological reactivity, and amino acid composition. Purified gp52 (sl) in radioimmunoprecipitation inhibition assays reacted in two distinct patterns. One pattern showed partial displacement of antibody which could be converted to the second, a complete displacement, by heating the antigen, presumably by exposing additional reactive determinants. Biologically, the patterns of major MMTV polypeptide expression in milk correlated with spontaneous mammary tumor incidence in different strains of mice, indicating that the sl antigen is group specific for MMTV or that several mouse strains contain the same virus type.

Defective retrovirus-like 30S RNA species of rat and mouse cells are infectious if packaged by type C helper virus.

RNA species with properties of defective retrovirus-like 30S RNA genomes have previously been detected in both rats and mice and in some rat and mouse retroviruses. Using cell lines which express high levels of this retrovirus-like RNA, we formed pseudotypes of the 30S RNAs with helper-independent type C viruses. A pseudotype virus complex containing a mouse 30S subunit was transmitted to rat cells, and a pseudotype virus complex containing a rat 30S subunit was transmitted to bat cells. In other transmission experiments, a rat 30S subunit was isolated in nonproducer bat cells without detectable expression of the helper-independent type C virus used to pseudotype it. The results provide further support for the retrovirus-like nature of the rat 30S subunit and provide evidence which supports the protovirus hypothesis proposed by Temin.

Functional analysis of the ligand binding site of EGF-receptor utilizing chimeric chicken/human receptor molecules.

The epidermal growth factor (EGF)-receptor is composed of an extracellular ligand-binding region connected by a single transmembrane region to the cytoplasmic kinase domain. In spite of its importance for understanding signal transduction, the ligand-binding domain of the EGF-receptor is not yet defined. We describe the identification of a major ligand-binding domain of the EGF-receptor by utilizing chimeras between the human EGF-receptor and the chicken EGF-receptor. This approach is based on the fact that murine EGF binds to the chicken EGF-receptor with 100-fold lower affinity as compared to the human EGF-receptor. Hence, the substitution of various domains of the chicken EGF-receptor by domains of the human EGF-receptor may restore the higher binding affinity towards EGF, characteristic of the human receptor. We show that chimeric chicken/human EGF-receptor, which contains domain III of the extracellular region of the human receptor, behaves like the human EGF-receptor with respect to EGF binding affinity and biological responsiveness. However, a chimeric chicken/human EGF-receptor containing domains I and II of the human receptor behaves like the chicken rather than the human EGF-receptor. Moreover, two different monoclonal antibodies which compete for the binding of EGF to EGF-receptor recognize specifically domain III of the human EGF-receptor. It is concluded that domain III which is flanked by the two cysteine-rich domains is a major ligand-binding domain of the EGF-receptor.

Friend strain of spleen focus-forming virus is a recombinant between ecotropic murine type C virus and the env gene region of xenotropic type C virus.

The spleen focus-forming virus (SFFV), a replication-defective murine leukemia virus that causes the rapid transformation of certain hematopoietic target cells, has acquired specific xenotropic viral genetic information not contained in Friend helper virus. In the current studies, it is shown that a cDNA that represents a xenotropic virus portion of SFFV detects genetic sequences derived from the env gene region of murine xenotropic virus. The significance of the acquisition of these xenotropic viral sequences by SFFV is discussed with regard to their possible role in the rapid leukemogenicity of SFFV, and an analogy is drawn between the formation of SFFV and the formation of the Kirsten and Harvey sarcoma viruses.

Identification of a 30S RNA with properties of a defective type C virus in murine cells.

A novel species of 30S RNA has been detected in a variety of mouse cell lines. The 30S RNA is specifically packaged by helper-independent type C viruses propagated in such cells. Nucleic acid hybridization detects no homology between the 30SRNA and the genomic RNA of helper-independent mouse type C viruses. The properties of the 30S RNA suggest that it is a defective endogenous mouse type C virus and that it is analogous to a previously described class of defective endogenous rat type C virus, which has been shown previously to be the progenitor of Kirsten and Harvey murine sarcoma viruses.

Separation of sarcoma virus-specific and leukemia virus-specific genetic sequences of Moloney sarcoma virus.

We have studied the nucleic acid sequences in nonproducer cells transformed by Moloney sarcoma virus or Abelson leukemia virus (two types of replication-defective, RNA-containing, viruses isolated by passage of Moloney leukemia virus in BALB/c mice). DNA probes from the Moloney leukemia in virus detect RNA in both Abelson virus-transformed nonproducer cells and Moloney sarcoma virus-transformed nonproducer cells. A sarcoma-specific cDNA, prepared from the Moloney sarcoma virus, has extensive homology to RNA found in heterologous nonproducer cells transformed by Moloney sarcoma virus, has little homology to RNA in cells producing Moloney leukemia virus, and no detectable homology to RNA in nonproducer cells transformed by the Abelson virus. By analogy to earlier data on avian and mammalian sarcoma viruses, these results suggest that the Moloney sarcoma virus arose by recombination between a portion of the Moloney leukemia virus genome and additional sarcoma-specific information, and indicate that the expression of this information in not essential for Abelson virus-mediated fibroblast transformation.

Cloning of three human tyrosine phosphatases reveals a multigene family of receptor-linked protein-tyrosine-phosphatases expressed in brain.

A human brainstem cDNA library in bacteriophage lambda gt11 was screened under conditions of reduced hybridization stringency with a leukocyte common antigen (LCA) probe that spanned both conserved cytoplasmic domains. cDNA encoding a receptor-linked protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), RPTPase alpha, has been cloned and sequenced. Human RPTPase alpha consists of 802 amino acids. The extracellular domain of 150 residues includes a hydrophobic signal peptide and eight potential N-glycosylation sites. This is followed by a transmembrane region and two tandemly repeated conserved domains characteristic of all RPTPases identified thus far. The gene for RPTPase alpha has been localized to human chromosome region 20pter-20q12 by analysis of its segregation pattern in rodent-human somatic cell hybrids. Northern blot analysis revealed the presence of two major transcripts of 4.3 and 6.3 kilobases. In addition to RPTPase alpha, two other RPTPases (beta and gamma), identified in the same screen, have been partially cloned and sequenced. Analysis of sequence comparisons among LCA, the LCA-related protein LAR, and RPTPases alpha, beta, and gamma reveals the existence of a multigene family encoding different RPTPases, each containing a distinct extracellular domain, a single hydrophobic transmembrane region, and two tandemly repeated conserved cytoplasmic domains.

Identification of a Src SH3 domain binding motif by screening a random phage display library.

A phage display library was constructed in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different phage, was screened with a glutathione S-transferase (GST) fusion protein containing the Src homology 3 (Src SH3) domain and a protein kinase A phosphorylation site (GST/PKA/Src SH3). A family of proline-rich sequences was isolated following four cycles of enrichment and amplification. Phage containing these sequences were shown to specifically bind to the GST/PKA/Src SH3 protein but not to GST/PKA only. A comparison of the inferred amino acid sequence of the different phage clones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 domain binding motif identified independently during an affinity screen of a lambda-lox mouse embryo cDNA library using a 32P-labeled Src SH3 protein fragment as the probe (Y. Ivashchenko, manuscript in preparation). Peptides based upon the 7-amino acid SH3 binding domain core motif displayed strong binding to both the Src and to the Fyn SH3 domains, but failed to bind to the SH3 domain of p21 Ras-GTPase-activating protein (Ras-GAP) and other proteins. We anticipate that further screening of the phage display library will be a useful tool for the rapid identification of additional SH3 domain binding sequences and will also help to establish the essential core motifs that define the specificity of interactions among the diverse proteins containing SH3 domains and those containing SH3 binding motifs.

Characterization and separation of viral DNA polymerase in mouse milk.

Two DNA polymerase with properties of viral RNA-directed DNA polymerase can be found in RIII mouse milk. One enzyme is the polymerase of type-C viruses; this enzyme prefers manganese to magnesium with poly(rA).oligo(dT) as synthetic template, is inhibited by specific sera, and has an apparent molecular weight of 70,000. Milk from BALB/c and NIH Swiss mice contain a vast predominance of this type-C enzyme. The other DNA polymerase from RIII mouse milk prefers magnesium to manganese, is not inhibited by type-C antipolymerase serum, and appears larger on gel chromatography than the type-C viral polymerase. Its presence in milk from RIII mice and absence from milk of mice with low content of mammary tumor virus correlates to the relative degree of type-B virus expression in these mice. The DNA polymerase of Mason-Pfizer monkey virus, isolated from a rhesus monkey breast tumor, also has a marked preference for magnesium with poly(rA).oligo(dT), is not immunologically related to primate type-C viruses, and appears larger than the gibbon type-C enzyme on gel chromatography.

Isolation of a new simian foamy virus from a spider monkey brain culture.

A syncytium-forming (foamy) virus was isolated from a spider monkey brain cell culture. Cytopathic effect was observed both in the brain culture and in human embryonic kidney cells. Neutralizing antibody was present in the sera of the spider monkey from whom the isolation was made. The virus was inhibited by 5-bromo-2-deoxyuridine (20 mug/ml), contained a ribonucleic acid-dependent deoxyribonucleic acid polymerase, and had an infectivity peak at 1.15 g/cm(3) in a sucrose density gradient. The virus passed through a 220-nm but not a 100-nm membrane filter, was chloroform sensitive, and was inactivated at 56 C in 30 min. Hemagglutinating and hemadsorption activity was not noted with a variety of erythrocytes. The virion was spherical, formed in the cytoplasm, and was 105 to 115 nm in diameter. Ring-shaped nucleoids, 45 to 50 nm in diameter, were associated with tubular profiles. The virus was not neutralized by sera prepared against known viruses, including simian foamy virus types 1 through 7, Mason-Pfizer monkey virus, and bovine syncytial and measles viruses. Sera from a rabbit hyperimmunized with the isolate and sera from 19 spider monkeys had neutralizing antibody to the isolate; however, these sera did not cross-react with simian foamy virus types 1 through 7. Neutralizing antibody to the isolate was not detected in sera from 16 humans, 9 rhesus monkeys, and 10 chimpanzees.

Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor).

The control of vascular endothelial and smooth muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical vein endothelial cells, express HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with 125I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.

All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor.

Activation of the epidermal growth factor (EGF) receptor kinase leads to autophosphorylation and to the phosphorylation of various cellular substrates. The three known autophosphorylation sites of EGF receptor are located at the carboxyl-terminal tail where they probably act to compete with and thus modulate substrate phosphorylation. Mutational analysis and microsequencing techniques have been used to localize and identify new autophosphorylation site(s) of the EGF receptor. We have compared the phosphopeptide maps of human EGF receptor, and two deletion mutants lacking 63 and 126 amino acids from the carboxyl-terminal tail with the phosphopeptide maps of HER/neu and a chimeric EGF receptor containing the carboxyl-terminal tail of HER2/neu. HER2/neu is highly homologous to the EGF receptor, and it probably functions as a growth factor receptor for as yet unidentified growth factor. On the basis of this analysis, we have concluded that all autophosphorylation sites of EGF receptor and HER2/neu are located in their carboxyl-terminal tails. Utilizing the EGF receptors with carboxyl-terminal deletions, we were also able to identify tyr1086 as an additional autophosphorylation site of EGF receptor. Direct microsequencing of a phosphorylated tryptic peptide from the human EGF receptor confirmed this assignment.