Recombinant retroviruses in transgenic mice.
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Animals↗
Biomedical subjects
I M Verma
Publications and source records attributed to I M Verma.
Proto-oncogene fos: an inducible multifaceted gene.
Proto-oncogene fos, which is expressed during cell growth and cell differentiation and development, is a multifaceted gene. The viral homolog, v-fos, was identified as the resident transforming gene of FBJ-murine osteosarcoma virus, which induces bone tumors in mice. Owing to an in-frame deletion during the biogenesis of the v-fos gene, the products of viral and cellular fos proteins differ at their C-termini. Despite different C-termini, both fos proteins are nuclear in their location and can transform fibroblasts in vitro. However, transformation by the c-fos gene requires removal of a 67-base pair sequence from the 3' noncoding domain. Proto-oncogene fos is a highly inducible gene in response to a variety of growth factors and differentiation-specific inducers. The expression of the fos gene is not modulated during the cell cycle.
Animals↗
Proto-oncogene fos: an inducible gene.
Proto-oncogene fos is a multifaceted gene, which is expressed during cell growth, cell differentiation, and development. The viral homologue, v-fos, was identified as the resident transforming gene of FBJ-murine osteosarcoma virus which induces bone tumors in mice. Due to an in-fram deletion during the biogenesis of the v-fos gene, the products of viral and cellular fos proteins differ at their C-termini. Despite different C-termini, both fas proteins are nuclear in their location, and can transform fibroblasts in vitro. However, transformation by c-fos gene requires removal of a 67 base pair sequence from the 3' non-coding domain. Proto-oncogene fos is a highly inducible gene in response to a variety of growth factors and differentiation-specific inducers. The transcriptional enhancer and the inducible element of the fos gene have been mapped along with sequences in the 3' non-coding domain which may influence the stability of the c-fos transcripts. The c-fos gene transcription is regulated by positively and negatively acting cellular factors.
Gene Expression Regulation↗
Structural features of growth factor receptors with oncogenic potential.
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Amino Acid Sequence↗
Identification of a transcriptional enhancer element upstream from the proto-oncogene fos.
Sequences upstream from the proto-oncogene fos were shown to be essential for its transcription. Transient expression of the chloramphenicol acetyl-transferase (CAT) gene linked to upstream sequences of the fos gene including its promoter reveals that sequences located 64 to 404 base pairs 5' to the fos cap site contain a typical transcriptional enhancer. Moreover, these enhancer sequences, which are strikingly conserved between mouse and human fos genes, coincide with a deoxyribonuclease I-hypersensitive site in the chromatin. The expression of the fos-CAT fusion genes was stimulated only two to three times by the fos inducer 12-0-tetradecanoyl phorbol-13-acetate. The fos enhancer does not appear to be tissue-specific.
Acetyltransferases↗
Rapid induction of the expression of proto-oncogene fos during human monocytic differentiation.
The differentiation into macrophages of the monocytic cell line U-937 or the monomyelocytic cell line HL-60 induced by phorbol esters is accompanied by a rapid induction of the expression of the proto-oncogene c-fos. Maximal levels of c-fos gene transcripts accumulate within 20-30 min following induction. By 2 hr, the c-fos-specific mRNA level drops 5 to 10 fold below that detected at 30 min after induction. The concentration of c-fos mRNA remains unchanged for the next 5 days, at which time over 99% of the viable precursor cells are converted to adherent macrophages. c-fos protein can be detected within 20 min of induction, reaching maximal levels by 40-60 min. Little or no c-fos protein is detected by 120-150 min after induction, even though c-fos mRNA remains detectable. Presence of cycloheximide leads to superinduction of c-fos mRNA transcripts. No c-fos gene transcripts are detected when the HL-60 cell line is induced with Me2SO to differentiate to granulocytes. A role for the c-fos protein is envisaged in the monocyte differentiation pathway.
Cell Differentiation↗
Removal of a 67-base-pair sequence in the noncoding region of protooncogene fos converts it to a transforming gene.
Transformation of fibroblasts by protooncogene fos (c-fos) requires the linkage of viral long terminal repeat (LTR) sequences and interruption of 3'-noncoding sequences. We have identified an A + T-rich stretch of 67 nucleotides, located 627-693 base pairs downstream from the coding domain and 123-189 base pairs upstream from the putative poly(A) addition site, removal of which confers transforming activity to the c-fos gene. A novel regulation of the expression of the c-fos gene is proposed, which may be functional in vivo to prevent the gene from becoming an oncogene.
Animals↗
Efficient insertion of genes into the mouse germ line via retroviral vectors.
We present a general strategy for the efficient insertion of recombinant retroviral vector DNA into the mouse germ line via infection of preimplantation mouse embryos. Transgenic mice were generated that harbor a replication-competent recombinant retrovirus (delta Mo + Py M-MuLV) that lacks the Moloney murine leukemia virus (M-MuLV)-type enhancer sequence in the long terminal repeat (LTR). Instead, the LTR contains an enhancer element that permits polyoma virus F101 to grow in undifferentiated F9 embryonal carcinoma cells. Expression studies in different tissues of animals transgenic for delta Mo + Py M-MuLV indicate possibilities to target and modulate expression of retroviral recombinants in mice via their LTR enhancer sequences. In addition, 16 transgenic mice were generated that harbor proviral DNA of a defective recombinant retrovirus carrying a mutant dihydrofolate reductase gene.
Animals↗
Induction of the proto-oncogene fos by nerve growth factor.
Nerve growth factor (NGF) causes the differentiation of PC12 cells to sympathetic neuron-like cells and also induces a rapid but transient expression of fos mRNA and protein. fos mRNA transcripts can be detected 5 min after the addition of NGF, are maximally abundant after 30 min, and then their levels decrease. fos protein synthesis parallels the expression of fos mRNA, and the induced fos proteins are located in the nucleus. cAMP, epidermal growth factor, the phorbol ester phorbol 12-myristate 13-acetate, and K+ depolarization also induce the fos gene. Growth of PC12 cells in the presence of dexamethasone, which induces differentiation into chromaffin-like cells, is not accompanied by fos expression. We propose that while fos gene induction is associated with the differentiation of PC12 cells to sympathetic nerve, its enhanced expression is primarily involved in the anabolic responses induced by NGF and many growth factors.
Animals↗
Proto-oncogene fos is expressed during development, differentiation, and growth.
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Adrenal Gland Neoplasms↗
Deletion of the gag region from FBR murine osteosarcoma virus does not affect its enhanced transforming activity.
Both FBJ murine osteosarcoma virus (FBJ-MSV) and FBR-MSV induce transformation in tissue culture and osteogenic sarcomas in mice. In tissue culture, however, FBR-MSV induces larger foci with a shorter latency than those induced by FBJ-MSV. Transformation is dependent on expression of the fos oncogene in FBJ-MSV and of a gag-fos fusion protein in FBR-MSV. We have determined that the gag sequences can be deleted from FBR-MSV without affecting the high transforming activity of this virus in comparison to FBJ-MSV. The resultant virus, designated FBJ/R-MSV, has a protein coding region that is half the size of that of FBR-MSV and about one-third smaller than that of FBJ-MSV. Thus, FBJ/R-MSV will provide a useful tool for studying the transforming activity of the fos oncogene.
Animals↗
Viral and cellular fos proteins are complexed with a 39,000-dalton cellular protein.
The structure of viral and cellular fos gene products and their association with a 39,000-dalton cellular protein (p39) were investigated by using antisera raised against synthetic peptides. The first peptide, termed M, corresponded to amino acids 127 to 152 of the v-fos sequence, a region which is identical in c-fos. The second peptide, termed V, corresponded to the nine C-terminal amino acids of v-fos; this region is not present in c-fos. Rabbit antisera were purified by affinity chromatography against their respective peptides before being used for immunoprecipitation. M peptide antisera precipitated p55v-fos and p55c-fos, whereas V peptide antisera precipitated only p55v-fos. This observation confirms the prediction from nucleotide sequence analysis that these proteins are distinct at their C termini. p39 was precipitated in association with p55v-fos and p55c-fos by M and V peptide antisera. However, V peptide antisera did not precipitate p39 from cells expressing p55c-fos, even though the presence of p39 in such cells was demonstrated with M peptide antisera. Denaturation of cell lysates completely abolished the precipitation of p39, whereas the precipitation of p55v-fos was unaffected. Taken together, the data demonstrate that p39 exists in a complex with p55.
Amnion↗
Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene.
We constructed several retroviruses which transduced a mutant dihydrofolate reductase gene that was resistant to methotrexate inhibition and functioned as a dominant selectable marker. The titer of dihydrofolate reductase-transducing virus produced by virus-producing cells could be increased to very high levels by selection of the cells in increasing concentrations of methotrexate. Helper virus-free dihydrofolate reductase-transducing virus was also generated by using a broad-host-range amphotropic retroviral packaging system. Cell lines producing helper-free dihydrofolate reductase-transducing virus with a titer of 4 X 10(6) per ml were generated. These retroviral vectors should have general utility for high-efficiency transduction of genes in cultured cells and in animals.
Animals↗
Expression of the c-fos gene and of an fos-related gene is stimulated by platelet-derived growth factor.
Complementary DNA clones of genes induced by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells were isolated; one such clone contains a domain having nucleotide sequence homology with the third exon of c-fos. This nucleotide sequence homology is reflected in the predicted amino acid sequences of the gene products. Under low stringency conditions, the mouse v-fos gene cross-hybridizes with the PDGF-inducible complementary DNA clone. However, the messenger RNA transcripts of mouse c-fos and the new fos-related gene can be distinguished by gel electrophoresis and by S1 nuclease analysis. Expression of the authentic c-fos gene is induced by PDGF and superinduced by the combination of PDGF and cycloheximide.
Amino Acid Sequence↗
Infectious and selectable retrovirus containing an inducible rat growth hormone minigene.
A growth hormone minigene carrying its natural promoter (237 nucleotides of chromosomal DNA) was stably propagated in a murine retrovirus containing hypoxanthine-guanine phosphoribosyltransferase as a selectable marker. Glucocorticoid and thyroid hormone inducibility was transferred with the growth hormone gene. Recombinant virus with titers of 10(6) per milliliter was recovered. This demonstration that retroviruses can be used to transfer a nonselectable gene under its own regulatory control enlarges the scope of retroviral vectors as potent tools for gene transfer.
Animals↗
Expression of a retrovirus encoding human HPRT in mice.
Transmissible retroviruses encoding human hypoxanthine phosphoribosyltransferase (HPRT) were used to infect mouse bone marrow cells in vitro, and the infected cells were transplanted into mice. Both active human HPRT-protein and chronic HPRT-virus production were detected in hematopoietic tissue of the mice, showing transfer of the gene. These results indicate the possible use of retroviruses for somatic cell therapy.
Animals↗
Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells.
In order to further define the mechanisms by which polypeptide growth factors regulate gene transcription and cellular growth, expression cloning techniques were used to select human epidermal growth factor (EGF) receptor complementary DNA clones. The EGF 3' coding domain shows striking homology to the transforming gene product of avian erythroblastosis virus (v-erbB). Over-expression of EGF receptors in A431 cell lines correlates with increased EGF receptor mRNA levels and amplification (up to 110 times) of the apparently singular EGF receptor gene. There appear to be three cytoplasmic polyadenylated RNA products of EGF receptor gene expression in A431 cells, one of which contains only 5' (EGF binding domain) sequences and is postulated to encode the secreted EGF receptor-related protein.
Base Sequence↗
Expression of cellular oncogenes in human malignancies.
Cellular oncogenes have been implicated in the induction of malignant transformation in some model systems in vitro and may be related to malignancies in vivo in some vertebrate species. This article describes a study of the expression of 15 cellular oncogenes in fresh human tumors from 54 patients, representing 20 different tumor types. More than one cellular oncogene was transcriptionally active in all of the tumors examined. In 14 patients it was possible to study normal and malignant tissue from the same organ. In many of these patients, the transcriptional activity of certain oncogenes was greater in the malignant than the normal tissue. The cellular fes (feline sarcoma) oncogene, not previously known to be transcribed in mammalian tissue, was found to be active in lung and hematopoietic malignancies.
Adenocarcinoma↗