PubMed Health⌕ Search

Biomedical subjects

D Stehelin

Publications and source records attributed to D Stehelin.

At least 109 records · Page 6Linked to original sources
PubMed1985

The four C-terminal amino acids of the v-erbA polypeptide are encoded by an intronic sequence of the v-erbB oncogene.

The genome of avian erythroblastosis virus (AEV), a defective acute leukemia retrovirus, carries two distinct cell-derived oncogenes in the structure 5' delta gag-erbA-erbB-delta env3'. The nucleotide sequence of the v-erbA gene was recently reported. In order to determine the boundary between the two adjacent oncogenes, the sequence of the v-erbA/v-erbB junction of AEV was compared to that of a recombinant lambda phage containing a chicken cellular sequence representing the 5' part of c-erbB. The four C-terminal amino acids of v-erbA are in fact encoded by a c-erbB intron-derived sequence thus demonstrating that the virus acquired a truncated c-erbA gene. Furthermore the 7 to 10 amino acid residues upstream from the 4 C-terminal amino acids mentioned above appeared to be derived from env-related sequences. The splice acceptor site at the beginning of the only open reading frame for v-erbB is also present and functional in c-erbB when expressed to generate a truncated EGF (epidermal growth factor) receptor. Thus AEV joins a truncated erbA gene to a truncated erbB gene through env-derived sequences and intronic sequences from c-erbB.

Alpharetrovirus↗
PubMed1985

Mapping of amplified c-myb oncogene, sister chromatid exchanges, and karyotypic analysis of the COLO 205 colon carcinoma cell line.

We have studied molecular and chromosomal details of cytogenetic status in a human tumor cell line COLO 205 that shows a stable, approximately tenfold amplification of the c-myb oncogene. The amplified copies of c-myb reside in two marker chromosomes that may have evolved from chromosome #6 by complex chromosomal rearrangements. No homogeneously staining regions can be discerned at the site of c-myb amplification. We suggest that c-myb was amplified in situ in a chromosomal segment (6q22-24) that became a part of the marker chromosome, possibly through isochromosome formation followed by duplication, and without the extrachromosomal intermediate form of double minute chromosomes. There is an enhanced frequency of sister chromatid exchanges at the site of amplified c-myb. These results are discussed in the context of models for gene amplification and oncogene activation.

Adenocarcinoma↗
PubMed1984

Sequencing the erbA gene of avian erythroblastosis virus reveals a new type of oncogene.

Avian erythroblastosis virus (AEV) contains two distinct oncogenes, erbA and erbB . The erbB oncogene, which is homologous to a portion of the epidermal growth factor receptor, is related to the src family of oncogenes and efficiently transforms erythroblasts, whereas erbA potentiates the effects of erbB by blocking the differentiation of erythroblasts at an immature stage. This "potentiator" was sequenced; the amino acid sequence deduced from it was clearly different from the sequences of other known oncogene products and was related to carbonic anhydrases. These enzymes participate in the transport of carbon dioxide by erythrocytes, the precursors of which are main targets of avian erythroblastosis virus. A src-related oncogene such as erbB in synergy with an activated specific cell-derived gene such as erbA can profoundly affect early erythroid differentiation.

Alpharetrovirus↗
PubMed1984

Nucleotide sequence of the human c-myc locus: provocative open reading frame within the first exon.

The nucleotide sequence of a HindIII-EcoRI DNA fragment, 8 kbp long, of a lambda recombinant containing the whole human c-myc gene has been deduced by the method of Maxam and Gilbert. This fragment encodes the complex c-myc locus and the sequence provides information relative to the 2.7 kb long c-myc transcript. It appears that although exons 2 and 3 would code for a 48-K protein homologous to the myc domain of the viral p110 gag-myc protein, the first exon, which has a large open reading frame ending with a stop codon just upstream from the donor splice site, could code on its own for a 20-K protein. Speculations about the role of that putative protein on the regulation of the expression of exons 2 and 3 are made.

Base Sequence↗
PubMed1984

The second oncogene mil of avian retrovirus MH2 is related to the src gene family.

The nucleotide sequence of a PstI fragment prepared from a cloned MH2 virus genome, pMH2-Hd, has been deduced using chemical and enzymatic methods. This fragment, 1862 nucleotides in length, starts with the gag gene, encodes the v-mil sequence and stops within the v-myc gene. This sequence shows that the v-mil gene is fused to the gag gene giving rise to a fused polyprotein of 98 000 daltons: 515 amino acids at the amino terminus would correspond to p10, p19, p27 and part of p12 determinants, 347 amino acids at the carboxy terminus correspond to the v-mil specific sequence. The mil protein shares homology with a number of onc proteins such as src, fes, fms, mos, yes, fps and erbB, as well as with the catalytic chain of the cAMP-dependent protein kinase. This PstI fragment also encodes the beginning of the myc gene which was integrated in MH2 along with the 3' end of the preceding intron placing an acceptor splice site in front of the used open reading frame. As deduced from the sequence, the MH2 myc protein is not identical to the MC29 myc protein. It differs at its amino terminus, which contains little or no gag determinants, depending on the ATG used to initiate translation.

Amino Acid Sequence↗
PubMed1984

Increased transcription of the c-myc oncogene in two methylcholanthrene-induced quail fibroblastic cell lines.

The expression of three c-onc genes (c-erb, c-myc, c-myb) was investigated in five cell lines established from fibrosarcomas induced with 20-methylcholanthrene (MCA) of Japanese quails. These cell lines showed low levels of the three c-onc genes, with the exception of two cell lines that accumulated moderate (MCAQ 1-4) and large amounts (MCAQ3-5) of c-myc RNA. Molecular cloning and restriction endonuclease analyses indicated that expression of c-myc in these two cell lines were not associated with detectable rearrangements in the c-myc locus, that the size of the c-myc transcript (2.7 kb) in MCAQ 3-5 was similar to that of the normal c-myc messenger RNAs (mRNA) and that the transcriptional activation observed in MCAQ 3-5 was not mediated by the LTR (long terminal repeat) of a proximate ALV (avian leukosis virus) provirus. Finally, when analysed with the restriction enzymes Msp I and Hpa II, the c-myc locus of MCAQ 3-5 and MCAQ 1-4 was found hypomethylated as compared with that of the other cell lines tested that show low levels of c-myc transcripts. Our results suggest that one of the ways methylcholantrene could mediate transformation is by inducing an abnormal regulation of the c-myc gene.

Animals↗
PubMed1984

Spatial and temporal pattern of cellular myc oncogene expression in developing human placenta: implications for embryonic cell proliferation.

We have analyzed staged human placentas by Northern, dot blot, and in situ hybridization to human c-myc probes. Placental RNA exhibits a stage-specific appearance of a 2.4 kb transcript of the c-myc gene. The frequency of this transcript varies 20 to 30 fold over the course of placental development, showing a peak at 4-5 weeks after conception, where the myc transcripts comprise about 0.05% by weight of the total placental mRNA. A clear decline in placental c-myc transcription is seen before the end of the first trimester of pregnancy. In situ hybridization to 125I-labeled myc probes demonstrates an unequal spatial distribution of myc transcripts in placental with particularly high expression in the cytotrophoblastic shell of early placenta. Labeling of placental explants with 3H-thymidine, the localization of myc transcripts to cytotrophoblasts, and the temporal pattern of myc expression all support a strong correlation between myc transcript abundance and cytotrophoblast proliferation. We argue for a role for the c-myc gene in the proliferation of normal cells in this tissue.

Cell Division↗
PubMed1983

Characterization of exogenous proviral sequences in hamster tumor cell lines transformed by Rous sarcoma virus rescued from XC cells.

Alterations in viral structural genes have been studied in five cell lines derived from Syrian hamster tumors which had been induced by the virus rescued from XC cells by transfection. Two cell lines, H-18 and H-20, have all the viral structural genes expressed, but a new EcoRI recognition site appeared in the region of the pol gene sequence. Provirus present in H-12 lacks the 3' part of the gag gene sequences as well as the pol gene, therefore, it gives rise to an anomalous 1.8 Md EcoRI fragment. This line also does not synthesize viral RNA of genomic size, and none of the subgenomic RNAs found hybridized with the DNApol probe. The H-19 cell line harbors only the src gene and LTR sequences, the U3 part of which seems incomplete or different from that of PR-RSV. The cryptic proviral structure in H-19 is transcribed into src mRNA. The degree of transcription of the src gene is about 25 viral RNA equivalents per cell. The H-9 cells harbor the complete provirus and, in addition, proviral structures having the deletion in gag-pol genes. The possible ways of development of provirus alterations and the role of cryptic proviral sequences in oncogenesis are discussed.

Animals↗
PubMed1983

Two different types of transcription for the myelocytomatosis viruses MH2 and CMII.

The four avian defective leukemia retroviruses (DLVs) MC29, CMII, MH2 and OK10 all transform primarily macrophages in an in vitro bone marrow transformation assay, and contain specific nucleotide sequences closely related to the myc gene of MC29. These viruses were thought to express their oncogenic potential through a gag-myc fusion polyprotein, since fusion polyproteins were found in all tested cells transformed by MC29. We show here that MH2 virus does not conform to this model. Whereas MC29 produces only one mRNA detectable by RNA blotting in productively transformed cells, we reported recently that OK10 induced the synthesis of two myc-containing mRNAs, the smaller species being a spliced mRNA and a possible candidate for a transforming protein lacking gag determinants. However, the studies with OK10 were ambiguous because this virus produced also, in infected cells, a fusion protein containing gag, pol and myc determinants. We have therefore investigated the transcription pattern of the two other members of this group of viruses, namely CMII and MH2. Our results show that CMII resembles MC29 whereas MH2 produces, as OK10, two mRNAs containing myc-related sequences. However, unlike OK10, the MH2 fusion protein of 100 kd described previously cannot contain myc determinants and thus is likely to produce from its subgenomic mRNA a v-myc protein-lacking gag determinants. We thus conclude that the product of the v-myc oncogene is transforming with (MC29) or without (MH2) its fusion to gag determinants and that the multiple oncogenic spectrum is not basically affected since MH2 and MC29 both transform macrophages, fibroblasts and epithelial cells.

Avian Leukosis Virus↗
PubMed1983

The human DNA locus related to the oncogene myb of avian myeloblastosis virus (AMV): molecular cloning and structural characterization.

Chicken and human cell DNA contains sequences homologous to the avian myeloblastosis virus oncogene, v-myb. These cellular sequences, c-myb (human) and c-myb (chicken), were isolated from libraries of human or chicken cell DNA fragments, generated by partial digestion with the restriction enzymes AluI and HaeIII, and compared. The chicken c-myb locus isolated from two distinct overlapping recombinant phages, contained five contiguous EcoRI fragments of 5.4, 1.1, 2.1, 2.2 and 9 kbp, accounting for all the bands seen with a v-myb probe in a complete EcoRI digest of chicken cellular DNA. Likewise, the screening of the human library yielded a recombinant phage hybridizing with the v-myb specific probe, that contained five EcoRI fragments of 2.8, 2.6, 2.0, 1.2 and 5.0 kbp (the last ending with an artificial EcoRI site, due to the construction of the library) belonging to the c-myb (human) locus. Probes using the EcoRI chicken DNA cloned fragments revealed corresponding contiguous EcoRI fragments in the human clone. Subsequent analyses of cellular polyadenylated mRNA extracted from human and chicken cells allowed the identification of single RNA species of 3.8 and 4.0 kb, respectively, as the representative transcripts of the c-myb locus in the two species. Thus, c-myb appears as a single locus in man and chicken, conserved with a similar structure in the two distantly related species. Our preparation of a specific human c-myb probe with an increased sensitivity on DNA/RNA blots should facilitate analyses concerning this gene in human normal or tumour cells or tissues.

Animals↗
PubMed1983

Molecular cloning of the avian acute transforming retrovirus MH2 reveals a novel cell-derived sequence (v-mil) in addition to the myc oncogene.

Mill-Hill-2 virus (MH2) proviral DNA was cloned from a transformed non-producer cell culture (MH2QB2) through insertion of randomly cut high mol. wt. cellular DNA in the lambdoid vector L47.1. Restriction analysis of a suitable recombinant phage by Southern DNA blotting and hybridization with different probes allowed us to characterize the genetic organization of the provirus and to identify a novel MH2-specific sequence of at least 1.1kbp. Such a sequence, for which we propose the name v-mil, from MilI-Hill-2 virus, is not homologous to v-myc, the previously described oncogene of MH2, nor to avian leukaemia virus-related sequences. Evidence is presented here that v-mil has a cellular counterpart (c-mil) phylogenetically conserved in birds and mammals, including man, and expressed as a single RNA species at least in some tissues. MH2 virus might thus be regarded, like avian erythroblastosis virus or E26, as another example of retroviruses having recombined with more than one cellular gene.

Journal Article↗
PubMed1983

The cellular oncogenes c-myc, c-myb and c-erb are transcribed in defined types of avian hematopoietic cells.

The possible role of normal chicken cellular sequences c-erb, c-myb and c-myc, together referred to as c-onc genes and related to the oncogenes of defective avian acute leukemia retroviruses (DLVs), was investigated by determining the accumulation of c-onc RNA in different avian cells an cell lines. Levels of c-myc and in some instances c-myb RNA are elevated in immature hematopoietic cells or cell lines from various lineages but more mature hematopoietic cells, as well as non-hematopoietic cells, contain only low levels. In contrast, the level of c-erb RNA is generally low, but high in a small number of normal bone marrow cells. The results indicate that the cellular homologues of the viral oncogenes are differentially expressed during hematopoiesis. They also indicate that the hypothesis that DLV target cells express their homologous c-onc genes might hold for c-erb, but is not valid in its simple form for c-myc and c-myb.

Animals↗
PubMed1982

Molecular cloning and characterization of the chicken DNA locus related to the oncogene erbB of avian erythroblastosis virus.

Chicken cell DNA contains sequences which are homologous to the avian erythroblastosis virus oncogene v-erb. These cellular sequences (c-erb) have been isolated from a library of chicken cell DNA fragments generated by partial digestion with AluI and HaeIII and shown to be shared by at least two loci in the chicken DNA. One of them, denoted c-erbB, contains approximately 1.8 kilobase pairs of chicken DNA homologous to the 3' part of the v-erb oncogene (v-erbB). Restriction mapping studies show that the c-erbB DNA sequences homologous to v-erbB are distributed among six EcoRI fragments located in a single genomic region. Heteroduplexes between v-erbB in viral RNA and cloned c-erbB DNA show that the chicken DNA sequences homologous to v-erbB are interrupted by 11 DNA sequences not present in the v-erb oncogene. We conclude from our data that the c-erbB locus might represent the cellular progenitor for the v-erbB domain of the v-erb oncogene.

Alpharetrovirus↗
PubMed1982

Subgenomic mRNA in OK10 defective leukemia virus-transformed cells.

OK10, a defective leukemia virus, is produced as a defective particle by so-called nonproducer transformed quail fibroblasts. OK10 defective viral particles contain an 8-kilobases (kb)-long genomic RNA, lack any detectable reverse transcriptase activity, and are not infectious. We studied the genetic content of OK10 RNA extracted from both virions and infected cells. As shown by RNA-cDNA hybridizations in stringent conditions, about 77% (6.4 kb) of the OK10 8.0kb RNA was related to avian leukosis viruses in the three structural genes gag, pol, and env, as well as in the c region. The remainder of the OK10 genome-encoding capacity (</=1.6 kb) was homologous to the MC29-specific transforming sequence myc(m) and therefore has been named myc(o). EcoRI restriction analysis of the OK10 integrated proviral DNA with different probes indicated the presence of only one provirus in the OK10 QB5 clone, which agreed with the gene order: 5'-gag-Deltapol-myc(o)-Deltaenv-c- 3'. Heteroduplex molecules formed between the viral OK10 8.0-kb RNA and the 6.8-kb SacI DNA fragment of the Prague A strain of Rous sarcoma virus confirmed that structure and indicated that the myc(o) sequence formed a continuous RNA stretch of 1.4 to 1.6 kb long between Deltapol and Deltaenv. We also examined the myc(o)-containing mRNA's transcribed in OK10-transformed cells. OK10-transformed quail fibroblasts (OK10 QB5) transcribed two mRNA species of 8.0 and 3.6 kb containing the myc(o) sequence. The genetic content of the 3.6-kb species made it a possible maturation product of the genome size 8-kb species by splicing out the gag and pol sequences. In OK10-transformed bone marrow cells (OK10 BM), a stable bone marrow-derived cell line producing OK10, the myc(o) sequence was found in four RNA species of 11.0, 8.0, 7.0, and 3.6 kb. Again, the genetic content of these mRNA's indicated that (i) the 3.6-kb species could be spliced out of the 8.0-kb-genome size mRNA and (ii) the 11.0-kb-long mRNA could represent a read-through of the OK10 provirus, the corresponding maturation product being, then, a 7.0-kb mRNA. The 7.0- and 3.6- kb mRNA's both contained the myc(o) sequence, but no sequences related to the gag or pol gene. In conclusion, whereas the myc sequences have been generally thought to be expressed through a gag-onc fusion protein, as for MC29 and CMII viruses, our experiments indicate that they could also be expressed as a non-gag-related product made from a subgenomic mRNA in the OK10-transformed cells.

Animals↗
PubMed1982

Characterization of c-lil, a chicken cellular sequence associated with a stock of B77 avian sarcoma virus.

Using biochemical methods, we have shown that a new specific sequence, v-lil, is associated with a given stock of B77 avian sarcoma virus (clone 9). We prepared a DNA complementary to v-lil sequences, using substractive hybridizations, and investigated the properties of this sequence. v-lil has a genetic complexity of ca. 2,000 nucleotides and is not present in various stocks of avian sarcoma virus, avian leukosis virus, or defective leukemia virus. v-lil is not associated with B77 avian sarcoma virus isolated from the original tumor and thus has been acquired by in vitro passage of the virus on chicken embryo fibroblasts. A search for the origin of the v-lil sequence among the DNAs of different avian species has shown that a similar sequence, c-lil, is present in normal chicken DNA (1 to 2 copies per haploid genome). c-lil is not highly conserved but is present in the DNA of all chickens from the genus Gallus. The c-lil sequence is transcribed at a low level (1 to 3 copies per cell) in normal chicken embryo fibroblasts. The biological function, if any, of v-lil or its cellular equivalent has yet to be determined.

Animals↗
PubMed1981

Characterization of the oncogene (erb) of avian erythroblastosis virus and its cellular progenitor.

Avian erythroblastosis virus (AEV) induces primarily erythroblastosis when injected intravenously into susceptible chickens. In vitro, the hematopoietic target cells for transformation are the erythroblasts. Occasional sarcomas are also induced by intramuscular injection, and chicken or quail fibroblasts can be transformed in vitro. The transforming capacity of AEV was shown to be associated with the presence of a unique nucleotide sequence denoted erb in its genomic RNA. Using a simplified procedure, we prepared radioactive complementary DNA (cDNAaev) representative of the erb sequence at a high yield. Using a cDNAaev excess liquid hybridization technique adapted to defective retroviruses, we determined the complexity of the erb sequence to be 3,700 +/- 370 nucleotides. AEV-transformed erythroblasts, as well as fibroblasts, contained two polyadenylated viral mRNA species of 30 and 23S in similar high abundance (50 to 500 copies per cell). Both species were efficiently packaged into the virions. AEV-transformed erythroblasts contained additional high-molecular-weight mRNA species hybridizing with cDNAaev and cDNA5' but not with cDNA made to the helper leukosis virus used (cDNArep). The nature and the role, if any, of these bands remain unclear. The erb sequence had its counterpart in normal cellular DNA of all higher vertebrate species tested, including humans and fish (1 to 2 copies per haploid genome in the nonrepetitive fraction of the DNA). These cellular sequences (c-erb) were transcribed at low levels (1 to 2 RNA copies per cell) in chicken and quail fibroblasts, in which the two alleged domains of AEV-specific sequences corresponding to the 75,000- and 40,000-molecular-weight proteins seemed to be conserved phylogenetically and transcribed at similar low rates.

Alpharetrovirus↗
PubMed1980

Cell transformation by avian defective leukaemia viruses.

A comparative study of seven independently isolated defective leukaemia viruses has been carried out. Phenotypic analysis of the chicken bone marrow cells transformed in vitro allowed the separation of these seven viruses into three groups based on the differentiation phenotype of the transformed cell. Nucleic acid hybridization studies revealed that these seven viruses had acquired cellular sequences. Interestingly, these studies also showed that the viruses within the same biological grouping had acquired related sequences. This indicates that viruses that have acquired the same or similar cellular sequences have very similar oncogenic capabilities. Analysis of proteins expressed in cells transformed by these viruses demonstrated that the cellular sequences were usually inserted within the gene for the viral core proteins, gag. Therefore the cellular sequences are expressed as a gag-related fusion protein which has an amino-terminal region derived from the gag gene and a carboxy-terminal half derived from the cellular sequences. Two exceptions to this are discussed. The general conclusion from these studies is that defective leukaemia viruses transform cells by virtue of acquired host cellular sequences. The ability of these viruses to transform cells and the target cell specificity of the transformation depends on these cellular sequences.

Alpharetrovirus↗