PubMed Health⌕ Search

Biomedical subjects

U R Rapp

Publications and source records attributed to U R Rapp.

At least 199 records · Page 11Linked to original sources
PubMed1985

Detection of the myristylated gag-raf transforming protein with raf-specific antipeptide sera.

The post-translational modifications of the gag-raf fusion proteins of the 3611 murine sarcoma virus (MSV) have been examined by inhibiting glycosylation with tunicamycin and by in vivo labeling with [3H]myristic acid. The results show that P75gag-raf is myristylated but not glycosylated and that P90gag-raf is glycosylated but not myristylated (and is now termed gP90gag-raf). gP90gag-raf expression appeared to become lost during passage of the transformed cells, and consequently does not appear to be necessary for the maintenance of transformation. raf-specific sera for detecting gag-raf fusion proteins have been obtained from synthetic peptides made from different regions of the predicted v-raf sequence. Immunoprecipitation of P75gag-raf with raf-specific sera directly confirmed the deduced v-raf sequence. The fact that P75gag-raf is both myristylated and precipitated by antiserum to a predicted carboxyl-terminal peptide of the v-raf gene established that the mature protein represents the entire coding region. The gP90gag-raf thus appears to be a glycosylated form of P75gag-raf specified by the gag sequences of the fusion protein, in analogy with Pr65gag and gPr80gag of murine leukemia viruses. Antiserum to the carboxyl-terminal P75gag-raf peptide was the most efficient in immunoprecipitation, and will be useful for detecting the product of the c-raf gene.

Animals↗
PubMed1985

MMC-E cells--origin and changes in karyotype accompanying malignant transformation.

Karyotype analysis of an established nontumorigenic cell line (MMC-E) indicated that the cells are of rat origin instead of mouse, as interpreted earlier. This cell line, now termed RE (rat epithelial) had a karyotype of 39,X,-5,-15,-?16,+t(3q11q) in the stem line. There was also one subline that had a karyotype of 40,X,-5,-15,-?16,+t(3q11q), +?t(5;?). Chromosome changes and frequency of sister chromatid exchanges (SCEs) were studied from seven malignantly transformed RE cell lines. These included cells transformed by Moloney murine sarcoma virus, the acute transforming retrovirus 3611-MSV, murine leukemia virus, or ethylnitrosourea. Changes in chromosomes #3 and #5 seemed to be associated with malignant transformation of RE cells. In addition to the t(3q11q) and monosomy for chromosome #5 seen in the parent cell line, monosomy for chromosome #3 and the t(5;?) were observed in all malignant cell lines. The latter changes either were absent (monosomy for chromosome #3) or present only occasionally [t(5;?)] in the parental cells. The results of SCEs showed that the malignantly transformed cell lines do not have increased frequency of SCEs as compared with that of the parental cell line.

Animals↗
PubMed1985

Immunoprecipitation of insulin receptors from cultured human lymphocytes (IM-9 cells) by antibodies to pp60src.

The family of tyrosine-specific protein kinases includes proteins encoded by retroviral oncogenes as well as receptors for insulin and several growth factors. Antibodies to pp60src, the protein encoded by the src oncogene of Rous sarcoma virus (RSV), can specifically immunoprecipitate affinity-labeled insulin receptors from cultured human lymphocytes (IM-9 cells). This precipitation is specifically inhibited by the src gene product purified from RSV-transformed rat cells. These observations provide evidence that there is structural homology between the insulin receptors and pp60src.

Cross Reactions↗
PubMed1985

Integration of transfected LTR sequences into the c-raf proto-oncogene: activation by promoter insertion.

A malignant cell line (clone S1) isolated after co-transfection of normal NIH3T3 DNA and Moloney leukemia virus long terminal repeat (Mo-LTR) sequences has previously been described to contain an activated c-raf oncogene. Here, we report the isolation by molecular cloning and the structural analysis of the LTR-activated c-raf gene. As shown by Southern blot and nucleotide sequence analyses, the transfected Mo-LTR sequences integrated into the 5th intron of the endogenous c-raf proto-oncogene. This intragenic LTR insertion led to the expression of high levels of LTR-U5-c-raf hybrid transcripts indicating an initiation of transcription from the Mo-LTR promoter. Transcriptional activation of c-raf is accompanied by the synthesis of large amounts of cytoplasmic c-raf protein. Immunoblot analysis suggests that the proteins encoded by the LTR-activated c-raf gene are truncated compared with the normal c-raf gene product(s). Our results indicate a promoter insertion mechanism of c-raf activation.

Animals↗
PubMed1985

Rapid induction of hemopoietic neoplasms in newborn mice by a raf(mil)/myc recombinant murine retrovirus.

3611 MSV, a raf oncogene-transducing murine retrovirus, induced fibrosarcomas in newborn mice after a latency of 4 to 8 weeks. In contrast, newly constructed recombinant murine retroviruses carrying the myc oncogene did not induce tumors before greater than or equal to 9 weeks. A combination of both oncogenes in an infectious murine retrovirus induced hematopoietic neoplasms in addition to less prominent fibrosarcomas and pancreatic acinar dysplasia 1 to 3 weeks after inoculation. The hematological neoplasms consisted of immunoblastic lymphomas of T- and B-lineage cells and erythroblastosis. Cell lines from these tumors could be readily established in culture in regular medium, whereas culture of cells from raf oncogene-induced tumors required the addition of interleukin 3. In parallel to the synergistic action of both oncogenes on hematopoietic cells in vivo, we found that raf oncogene-induced transformation of fibroblast cell lines in culture was enhanced by the addition of myc, which by itself did not morphologically transform these permanent cell lines. We conclude that concomitant expression of raf and myc oncogenes in hematopoietic cells and fibroblastic cell lines enhances their respective transforming activities.

Animals↗
PubMed1985

Structure and biological activity of human homologs of the raf/mil oncogene.

Two human genes homologous to the raf/mil oncogene have been cloned and sequenced. One, c-raf-2, is a processed pseudogene; the other, c-raf-1, contains nine exons homologous to both raf and mil and two additional exons homologous to mil. A 3' portion of c-raf-1 containing six of the seven amino acid differences relative to murine v-raf can substitute for the 3' portion of v-raf in a transformation assay. Sequence homologies between c-raf-1 and Moloney leukemia virus at both ends of v-raf indicate that the viral gene was acquired by homologous recombination. Although the data are consistent with the traditional model of retroviral transduction, they also raise the possibility that the transduction occurred in a double crossover event between proviral DNA and the murine gene.

Amino Acid Sequence↗
PubMed1984

Primary structure of v-raf: relatedness to the src family of oncogenes.

A replication-defective, acute transforming retrovirus (murine sarcoma virus 3611) was isolated from mouse and molecularly cloned. The nucleotide sequence of 1.5 kilobases encompassing the transforming gene (v-raf) was determined. This sequence, which predicts the amino acid sequence of a gag-raf fusion protein, terminates 180 nucleotides from the 3' end of the acquired cellular sequence. Comparison of the predicted amino acid sequence of v-raf with the predicted amino acid sequences of other oncogenes reveals significant homologies to the src family of oncogenes. There is a lack of homology within the sequence of the tyrosine acceptor domain described for the phosphotyrosine kinase members of the src family of transforming proteins. Phylogenetic arrangement of this family of oncogenes suggests that tyrosine-specific phosphorylation may be a recently acquired activity.

Amino Acid Sequence↗
PubMed1984

Transformation of cultured epithelial cells by ethylnitrosourea. Altered expression of type I procollagen chains.

Cultured epithelial rodent cells were transformed in vitro using ethylnitrosourea as a carcinogen either alone or in combination with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The frequency of transformation in the absence of TPA was 5 X 10(-4) at 10 micrograms/ml ethylnitrosourea. Growth of ethylnitrosourea-treated cells in TPA-substituted medium increased the transformation frequency 8-fold. Colonies of transformed cells were isolated from soft agar and analyzed for the production of pericellular matrix glycoproteins. The ethylnitrosourea-transformed cells retained pericellular matrix structures, typical of the nontransformed control cells. Parent cells produced into their culture media fibronectin and procollagen types I and III as their major pericellular glycoproteins. The ethylnitrosourea-transformed cells synthesized and secreted altered procollagen polypeptides. The procollagen of ethylnitrosourea-transformed cells apparently consisted mainly of homotrimeric pro alpha 1 molecules, with smaller amounts of basement membrane procollagen-like chains. Fibronectin synthesis or secretion was not affected by ethylnitrosourea-induced transformation, but the production of fibronectin was enhanced in the transformed cultures treated with TPA. Also, the deposition of procollagen and fibronectin into the pericellular matrix was not affected by ethylnitrosourea-transformation. Very similar changes had previously been observed in murine sarcoma virus-transformed cells. The change of procollagen type I thus appears to be a correlate of malignant transformation of cultured epithelial cells. The results indicate that ethylnitrosourea can induce malignant transformation of epithelial cells in culture and modify production and deposition of pericullular glycoproteins.

Animals↗
PubMed1984

The human homologs of the raf (mil) oncogene are located on human chromosomes 3 and 4.

Two human genes that are homologous to both the murine transforming gene (oncogene) v-raf and the chicken transforming gene v-mil have been mapped by means of human-rodent somatic cell hybrids to human chromosomes previously devoid of known oncogenes. One gene, c-raf-2, which appears to be a processed pseudogene, is located on chromosome 4. The other gene, c-raf-1, which appears to be the active gene, is located on chromosome 3 and has been regionally mapped by chromosomal in situ hybridization to 3p25. This assignment correlates with specific chromosomal abnormalities associated with certain human malignancies.

Adenocarcinoma↗
PubMed1984

A new oncogene, c-raf, is located on mouse chromosome 6.

The recently described acute transforming virus 3611-MSV contains cellular sequences designated v-raf. Mouse cellular DNA contains a single-copy sequence homologous to this oncogene (c-raf), and Southern blot analysis of hamster-mouse somatic cell hybrid DNAs showed that the mouse c-raf sequence is present on chromosome 6.

Animals↗
PubMed1984

Envelope gene sequence of two in vitro-generated mink cell focus-forming murine leukemia viruses which contain the entire gp70 sequence of the endogenous nonecotropic parent.

The mink cell focus-forming (MCF) class of recombinant murine leukemia viruses (CI-1 to 4) were isolated from iododeoxyuridine-induced C3H/MCA 5 cells in culture and molecularly cloned. These genomes included infectious (CI-3) and defective (CI-4) recombinants. A total of 2,408 nucleotides of CI-3 virus DNA, including the MCF envelope gene, were sequenced and compared with ecotropic, dual-tropic, and xenotropic sequences. The extent of recombinational exchange in CI-3 was from 145 nucleotides 3' of the splice acceptor site for the envelope mRNA to nucleotide 1,722, between the end of gp70 and the beginning of Prp15E. Thus, the entire gp70 sequence of the endogenous nonecotropic parent was present in this recombinant. The nature and location of the recombinant junctions were consistent with a mechanism involving DNA exchange during reverse transcription. Comparison of the substituted sequence in CI-3 with that of Moloney MCF virus suggests a very close relationship, if not identity, between the endogenous dual-tropic proviruses from which they were derived. A nonidentity of xenotropic and MCF gp70s was observed, suggesting that xenotropic murine leukemia viruses are not the nonecotropic parent of the env gene of MCF murine leukemia viruses. The replication-defective virus CI-4 had a 684-nucleotide deletion present in the env gene, eliminating the hydrophobic regions within the gp70 carboxy end and the p15E amino end. This sequence was bordered by an 11-nucleotide direct repeat in CI-3 viral DNA.

Amino Acid Sequence↗
PubMed1984

3'-Terminal region of avian carcinoma virus MH2 shares sequence elements with avian sarcoma viruses Y73 and SR-A.

We determined the nucleotide sequence of the acute transforming avian retrovirus MH2 from an HgiAI site within the coding region of its oncogene, v-myc, to the KpnI site within the long terminal repeat. Comparison with published sequences from other retroviruses allowed us to identify all sequence elements in this region. We conclude that MH2 contains a unique assembly of 3'-terminal sequences, which includes part of the helper virus-derived SPC region of avian sarcoma virus Y73 and the complete F3 and F1 segments of Rous sarcoma virus strain SR-A.

Animals↗
PubMed1983

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar.

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.

Agar↗
PubMed1983

Activation of a type C virus particle in cells from the inbred mouse strain 129/J: antigenic relationship with the horizontally transmitted type C viruses of primates. Brief report.

We have activated a thymus-derived fibroblast cell line from 129/J mice for production of noninfectious type C viral particles. Retroviral particles were produced continuously after cells were treated with ethylnitrosourea, a powerful mutagen and carcinogen. Antigenic analysis of the major structural protein of these particles showed a close relationship with an infectious virus from Mus caroli, a potential progenitor of the horizontally transmitted primate type C viruses. This finding demonstrates the potential of cells from inbred strains of Mus musculus to express upon mutagenesis antigens of the Mus caroli class of endogenous MuLV.

Animals↗
PubMed1983

Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus.

We have molecularly cloned a unique acutely transforming replication-defective mouse type C virus (3611-MSV) and characterized its acquired oncogene. The viral genome closely resembles Moloney (M) murine leukemia virus (MuLV), except for a substitution in M-MuLV in the middle of p30 and the middle of the polymerase gene (pol). Heteroduplex analysis revealed that 2.4 kilobases of M-MuLV DNA were replaced by 1.2 kilobases of cellular DNA. The junctions between viral and cellular sequences were determined by DNA sequence analysis to be 517 nucleotides into the p30 sequence and 1,920 nucleotides into the polymerase sequence. Comparison of the transforming gene from 3611-MSV, designated v-raf, with previously isolated retrovirus oncogenes either by direct hybridization or by comparison of restriction fragments of their cellular homologs shows it to be unique. Transfection of NIH 3T3 cells with cloned 3611-MSV proviral DNA leads to highly efficient transformation and the recovered virus elicits tumors in mice typical of the 3611-MSV virus. Transfected NIH 3T3 cells express two 3611-MSV-specific polyproteins (P75 and P90), both of which contain NH2-terminal gag gene-encoded components linked to the acquired sequence (v-raf) translational product. The cellular homolog, c-raf, is present in one or two copies per haploid genome in mouse and human DNA.

Animals↗
PubMed1983

Genome structure of mink cell focus-forming murine leukemia virus in epithelial mink lung cells transformed vitro by iododeoxyuridine-induced C3H/MuLV cells.

We characterized mink cell focus-forming murine leukemia viruses that were isolated from C3H/MCA-5 cells after induction with 5-iododeoxyuridine in culture. Mink lung epithelial cells malignantly transformed in vitro by induced virus were the source of four molecular clones of mink cell focus-forming virus. CI-1, CI-2, CI-3, and CI-4. Three clones, CI-1, CI-2, and CI-3, had full-length mink cell focus-forming viral genomes, one of which (CI-3) was infectious. In addition, we obtained a defective viral genome (CI-4) which had a deletion in the envelope gene. A comparison between the envelope genes of CI-4 and those of spleen focus-forming virus by heteroduplex mapping showed close homology in the substitution region and defined the deletion as being identical to the p15E deletion of spleen focus-forming virus. The recombinant mink cell focus-forming genomes are not endogenous in C3H/MCA-5 cells and therefore must have been formed in culture after induction by 5-iododeoxyuridine. CI-3, the infectious clone of mink cell focus-forming murine leukemia virus, was dualtropic, and mink cells infected with CI-3 were altered in their response to epidermal growth factor. In the presence of epidermal growth factor at 10 ng/ml, uninfected mink cells retained their epithelial morphology in monolayer culture and did not form colonies in soft agar. In contrast, CI-3 virus-infected mink cells grew with fibroblastic morphology in monolayer culture and showed an increased growth rate in soft agar in the presence of epidermal growth factor.

AKR murine leukemia virus↗
PubMed1983

Kinetics of expression of infectious ecotropic, xenotropic, and mink cell focus-forming murine leukemia virus after 5-iododeoxyuridine induction of cells from high- and low-leukemia mouse strains.

Endogenous murine leukemia virus (MuLV) was induced with 5-iododeoxyuridine (IdUrd) from the high-leukemia mouse strain AKR and from two low-leukemia strains, C3H/He and BALB/c. A virus-free cell line from strain AKR readily gave rise to infectious, XC-positive MuLV upon treatment with IdUrd, whereas cells from strains C3H/He and BALB/c produced replication-deficient, XC-negative MuLV. IdUrd-induced cells also produced xenotropic and mink cell focus-forming MuLV. Xenotropic virus emerged at a higher titer from both AKR and BALB/c cells during two discrete time intervals, first at day 3 after induction and a second time during spread of the induced ecotropic MuLV. Xenotropic and mink cell focus-forming MuLVs were also produced by IdUrd-induced C3H/He cells but required another round of infection in Sc-1 cells for detection. The in vitro infectivity of endogenous ecotropic MuLV isolated by IdUrd induction from C3H/He cells correlated with pathogenicity in the Fv-1-compatible, leukemia-negative mouse strain NFS/N. Thus, the virulence of endogenous ecotropic MuLV may be an important factor in determining the leukemia incidence in these inbred strains of mice.

AKR murine leukemia virus↗