Expression of cellular oncogenes.
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Animals↗
Biomedical subjects
I M Verma
Publications and source records attributed to I M Verma.
FBR murine osteosarcoma virus. I. Molecular analysis and characterization of a 75,000-Da gag-fos fusion product.
The FBR murine osteosarcoma virus complex induces bone tumors with a similar latency and pathology to those induced by the FBJ virus complex. FBR murine sarcoma virus ( FBR -MSV) has been isolated from its helper virus(es) by the establishment of transformed nonproducer cells. These cells were found to express a 75,000-Da protein (P75) which was antigenically related to the p55 oncogene product of the FBJ murine osteosarcoma virus ( FBJ -MSV). P75 also contained antigenic determinants of murine leukemia virus (MLV) gag gene p15, p12, and p30 proteins, and is therefore a gag- fos fusion protein ( P75gag - fos ). P75gag - fos is a phosphoprotein and is found primarily in the nucleus. Only a single species of RNA, of 3.3 kb, was identified in FBR -MSV-transformed nonproducer cells using both fos and MLV probes, which suggested that P75gag - fos was expressed from genome-sized RNA. Chromosomal DNA from one nonproducer cell line was found to contain a single EcoRI restriction fragment of 12 kb pairs (kbp) which encompassed the FBR -MSV provirus. This DNA fragment was molecularly cloned into bacteriophage Charon 30 (lambda FBR -1), and a 7.5-kbp HindIII restriction fragment containing the entire provirus was subsequently subcloned into pBR322 ( pFBR -1). DNA from pFBR -1 was capable of inducing morphological transformation of mouse and rat fibroblasts in tissue culture. In addition, transfected cells expressed the FBR -MSV P75gag - fos protein.
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FBR murine osteosarcoma virus. II. Nucleotide sequence of the provirus reveals that the genome contains sequences acquired from two cellular genes.
The complete nucleotide sequence of the FBR proviral DNA has been determined. The provirus of 3791 nucleotides (specifying a genome of 3284 bases) encodes a single gag- fos fusion product of 554 amino acids. The fos portion of the gene lacks the sequences which code for the first 24 and the last 98 amino acids of the 380-amino acid mouse c- fos gene product. In addition, the coding region has sustained three in-frame deletions, one in the p30gag portion, and two in the fos region, as compared to the sequences of AKR-MLV and the c- fos gene, respectively. The gene product terminates in sequences, termed v-fox, that are present in uninfected mouse DNA at loci unrelated to the c- fos gene. The c-fox gene(s) is expressed as an abundant class of polyadenylated RNA in normal mouse tissues.
Amino Acid Sequence↗
c-fos protein can induce cellular transformation: a novel mechanism of activation of a cellular oncogene.
The FBJ murine osteosarcoma virus (FBJ-MuSV) induces tumors in vivo and transformation in vitro. Transformation is due to the expression of a single viral protein (p55v-fos) which is encoded by sequences derived from mouse genetic material. The homologous cellular gene (c-fos) does not transform cultured cells after introduction by transfection. We show that even though the c-fos protein is completely different from the v-fos protein at its C terminus, it is capable of transforming cultured fibroblasts. However, activation of the transforming potential of the c-fos gene requires two manipulations--a transcriptional enhancer sequence must be linked to the gene and an interaction at the 3' end of the gene, which inhibits transformation, must be disrupted. Our studies show that normal cellular protein can induce transformation when expressed in an inappropriate cell type.
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Viral and cellular fos proteins: a comparative analysis.
The FBJ murine osteosarcoma virus (FBJ-MuSV) induces osteosarcomas in mice and transforms fibroblasts in vitro. It contains an oncogene termed v-fos derived from a normal cellular gene by recombination with an associated helper virus. The product of the v-fos gene is a 55,000 dalton protein, p55v-fos. This protein was found in the nuclei of cells containing amplified levels of the v-fos gene, and also in the nuclei of virus-transformed cells. The c-fos protein was localized in the nuclei of normal mouse amnion cells and in the nuclei of cells transformed by a recombinant plasmid that expresses the c-fos gene product. However, p55c-fos undergoes more extensive post-translational modification in the nucleus than p55v-fos. Immunofluorescence data indicate that the level of p55c-fos in normal mouse amnion cells is similar to that found in fibroblasts transformed by the v-fos or c-fos proteins.
Amnion↗
Human c-fos oncogene mapped within chromosomal region 14q21----q31.
The human cellular homolog (c-fos) of the transforming gene of Finkel-Biskis-Jinkins (FBJ) murine osteosarcoma virus was mapped to a single human chromosome. DNA from a series of 31 mouse-human somatic cell hybrid lines was probed with v- and c-fos molecular clones by Southern blotting. Human c-fos segregated with the distal region of the long arm of human chromosome 14. In situ hybridization of 125I-labeled human c-fos probe to normal human metaphase chromosomes independently confirmed these results and localized the c-fos oncogene to region 14q21----q31.
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Two base changes restore infectivity to a noninfectious molecular clone of Moloney murine leukemia virus (pMLV-1).
The complete nucleotide sequence of a molecular clone of Moloney murine leukemia virus (pMLV-1) has previously been reported (Shinnick et al., Nature [London] 293:543-548, 1981). However, pMLV-1 does not generate infectious virus after transfection into cells (Berns et al., J. Virol. 36:254-263, 1980). The lesion in pMLV-1 has been localized by determining the biological activity of recombinants containing DNA from an infectious clone of Moloney murine leukemia virus (pMLV-48) and pMLV-1. Replacement of a 1.0-kilobase pair region which spans the gag-pol junction of pMLV-1 with the corresponding DNA fragment from the infectious clone restores its infectivity. Nucleotide sequence analysis of this fragment obtained from the infectious clone (pMLV-48) and pMLV-1 reveals two single base pair changes, one in the p30gag gene and the other in the 5' end of the pol gene. The mutation in the pol gene does not affect the production of infectious virus but renders them XC negative, whereas the mutation in the gag gene appears to be lethal. The complete nucleotide sequence of an infectious clone of Moloney murine leukemia virus can now be deduced.
Base Sequence↗
Elements in the long terminal repeat of murine retroviruses enhance stable transformation by thymidine kinase gene.
We have investigated the effects of long terminal repeats (LTRs) of murine retroviruses on the frequency of obtaining stable transfectants by the herpes virus thymidine kinase (TK) gene. The results indicate that addition of LTRs enhances the number of TK+ transformants by 10-20 fold. A 5-12 fold enhancement was also observed when chromosomal DNA from either human or hamster cells was mixed with a plasmid containing LTR sequences and transfected onto LTR- cells. The LTR sequences involved in the enhancement were localized in the region which contains tandem repeats. All other regions of the LTR did not show any enhancement of stable TK+ transfectants. The location or the orientation of the enhancer sequences with respect to the TK gene did not exert any influence on the frequency of transformation. The enhancement effect does not appear to be linked to either increased numbers of chromosomal integrations or elevated levels of transcription of the TK gene.
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Expression of c-onc genes: c-fos transcripts accumulate to high levels during development of mouse placenta, yolk sac and amnion.
The c-onc genes c-fos and c-fms are expressed at high levels specifically in mouse extra-embryonal tissues. Here, we report the results of a detailed analysis of expression of these genes within the developing placenta and extra-embryonal membranes (i.e., yolk sac and amnion). (i) The c-fos gene is expressed at relatively high, but nearly constant levels in the undissected placenta throughout gestation. (ii) The level of c-fos transcripts is greater than or equal to 15-fold higher in the separated outer portion of the midgestation placenta (primarily undifferentiated fetus-derived cytotrophoblast maternal decidua) relative to the inner moiety (predominantly differentiated syncytiotrophoblast). (iii) In the inner placenta and in the extra-embryonal membranes c-fos transcripts accumulate as gestation proceeds. The abundance of c-fos transcripts in the micro-surgically isolated 18th day amnion reaches a level which is two orders of magnitude greater than that in midgestation fetuses, and is thus close to the level of v-fos transcripts in virus-transformed cells. (iv) The distribution of c-fos transcripts within the developing extra-embryonal tissues is markedly different from that of the c-fms gene. It is suggested that the c-fos and c-fms proteins may participate in differentiation, growth or transport processes occurring in mouse extra-embryonal tissues.
Amnion↗
Expression of FBJ-MSV oncogene (fos) product in bacteria.
The protein predicted from the DNA sequence of the FBJ murine osteosarcoma virus (FBJ-MSV) onc gene (v-fos) was expressed in Escherichia coli under the control of the tryptophan operon regulatory region. The 381-amino acid protein was identified by synthesis in minicells isolated from bacteria containing the expression vector plasmid. A 52,000-Da protein was made in these minicells, along with the proteins encoded by the beta-lactamase gene present on the expression vector plasmid. Synthesis of the 52K protein was repressed in trpR+ E. coli minicells in the presence of tryptophan, whereas the protein was synthesized under the same conditions in trpR- (derepressed) minicells. The beta-lactamase proteins, however, were synthesized under both conditions. The synthesis of the viral protein, therefore, was directed by the trp operon promoter. The tryptic peptide map of the 52K bacterial protein labeled with [35S]methionine was compared to that of the 35S-labeled protein immunoprecipitated from FBJ-MSV transformed rat cells using tumor-bearing rat sera. The peptide map of the p55 protein, previously identified as a candidate transforming protein in FBJ-MSV transformed cells, matches exactly that of the bacterial 52K protein. The eukaryotic cell protein, however, differs slightly in electrophoretic mobility in SDS gels, most likely due to post-translational modification which does not occur in bacteria.
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Analysis of FBJ-MuSV provirus and c-fos (mouse) gene reveals that viral and cellular fos gene products have different carboxy termini.
The complete nucleotide sequence of the FBJ-MuSV proviral DNA and the cellular homolog (c-fos) of its oncogene (v-fos) have been determined. The 4026 nucleotide long FBJ-MuSV proviral DNA contains two long terminal repeats, a substitution of 1639 nucleotides of mouse cellular DNA (v-fos) and the 3' end of the env gene derived from FBJ-MuLV. The sequences of the parental FBJ-MuLV and the cellular c-fos (mouse) gene share five of five nucleotides at the 5' end and ten of 11 nucleotides at the 3' end of the v-fos substitution. When compared with the v-fos sequences, the c-fos gene contains four discontinuous regions, three of which are flanked by sequences characteristic of introns. Direct sequence analysis of c-fos (mouse) RNA by primer extension demonstrates that the fourth discontinuity is due to a 104 bp deletion in the v-fos gene. As a consequence of the deletion, the predicted v-fos and c-fos gene products differ at their C termini.
Amino Acid Sequence↗
Complete nucleotide sequence of a human c-onc gene: deduced amino acid sequence of the human c-fos protein.
The complete nucleotide sequence of the c-fos(human) gene, the human cellular homolog of the oncogene (v-fos) of Finkel-Biskis-Jinkins murine osteosarcoma virus, has been determined. The c-fos(human) gene contains four discontinuous regions when compared with the v-fos gene. Three of the discontinuities are flanked by sequences characteristic of introns, while the fourth discontinuity is due to a deletion of 104 base pairs in the v-fos gene. As a consequence of the deletion, the predicted c-fos(human) and v-fos gene products differ at their carboxyl termini. Transcripts of 2.2 kilobases from the c-fos(human) gene have been identified in human cells. The sizes of these transcripts are in close agreement with the size expected from the nucleotide sequence after removal of introns.
Amino Acid Sequence↗
A transmissible retrovirus expressing human hypoxanthine phosphoribosyltransferase (HPRT): gene transfer into cells obtained from humans deficient in HPRT.
A cDNA corresponding to the human gene for hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) has been ligated into murine retroviral vectors such that it is under the transcriptional control of viral long terminal repeats. Transfection of HPRT- cells followed by superinfection with various helper viruses has led to the rescue of chimeric virus capable of transmitting the HPRT+ phenotype to HPRT- rodent or human cells. These genetically transformed cells contain authentic human HPRT at levels similar to normal HPRT+ cells.
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Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells.
The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human).
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Transcription of c-onc genes c-rasKi and c-fms during mouse development.
We investigated the expression of cellular sequences c-rasKi and c-fms, which are homologous to the oncogenes of Kirsten rat sarcoma virus and the McDonough strain of feline sarcoma virus, during murine development and in a variety of mouse tissues. The c-rasKi gene was found to be transcribed into two mRNA species of approximately 2.0 and 4.4 kilobases, whereas a single c-fms-related transcript of approximately 3.7 kilobases was identified. The c-rasKi gene appeared to be expressed ubiquitously, since similar levels of transcripts were observed in embryos, fetuses, extraembryonal structures, and a variety of postnatal tissues. In contrast, significant expression of c-fms was found to be confined to the placenta and extraembryonal membranes (i.e., combined yolk sac and amnion). The concentration of c-fms transcripts in the placenta increased approximately 15-fold (relative to day-7 to day-9 conceptuses) during development before reaching a plateau at day 14 to 15 of gestation. The time course of cfms expression in the extraembryonal membranes appeared to parallel the stage-specific pattern observed in the placenta. The level of c-fms transcripts in the extraembryonal tissues reached a level which was approximately 20- to 50-fold greater than that in the fetus. These findings suggest that the c-fms gene product may play a role in differentiation of extraembryonal structures or in transport processes occurring in these tissues. Our results indicate that the c-onc genes analyzed in the present study exert essentially different functions during mouse development.
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Differential expression of cellular oncogenes during pre- and postnatal development of the mouse.
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Age Factors↗
Moloney murine leukemia virus-induced tumors: recombinant proviruses in active chromatin regions.
The DNase I sensitivity of chromosomal DNA regions carrying integrated proviral genomes of Moloney (M-MuLV) and AKR Murine Leukemia Virus (AKR-MuLV), and the cellular homologue of the mos-gene (c-mos) of Moloney Sarcoma Virus (MSV) were studied in tumor tissues of leukemic mice. The genetically transmitted sequences of M-MuLV, AKR-MuLV, and the c-mos gene are all in DNase I resistant chromatin conformations in M-MuLV-induced tumors. Each M-MuLV-induced tumor contained at least one somatically acquired integrated recombinant MuLV genome that displayed two main characteristic features of active chromatin: a) a configuration hypersensitive to DNase I, and b) extensive hypomethylation. DNase I hypersensitive sites were mapped at the junction of cellular sequences and the 5'-viral large terminal repeat (LTR). Expression of a recombinant MuLV seems therefore to be a necessary feature to maintain the transformed state.
AKR murine leukemia virus↗
Detection of a transforming gene product in cells transformed by Moloney murine sarcoma virus.
We identified, in cells transformed by Moloney murine sarcoma virus (M-MuSV clone 124), a protein encoded by the M-MuSV transforming gene, v-mos. An antiserum against a synthetic peptide corresponding to the C terminus of a protein predicted from the v-mos nucleotide sequence specifically recognizes a protein doublet of approximately 37,000 daltons from 35S-methionine-labeled M-MuSV 124-transformed producer cells. By peptide mapping, this protein is almost identical to the 37 kd in vitro translation product from the M-MuSV v-mos gene. Immunoprecipitates from 32P-labeled cells contain a single v-mos-specific phosphoprotein, which has at least six sites of phosphorylation containing phosphoserine. Pulse-chase experiments show that the lower band in the 35S-methionine-labeled doublet is the primary translation product, which is modified, probably by phosphorylation, to yield the upper band. A similar mos protein is immunoprecipitated from HT1-MuSV-transformed cells, but not from uninfected NIH/3T3 cells. These mos proteins are present at very low levels in transformed cell lines. Cells acutely infected with M-MuSV 124, however, transiently contain much higher levels of the mos protein. These high levels coincide with extensive cell mortality.
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