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D Stehelin

Publications and source records attributed to D Stehelin.

At least 73 records · Page 4Linked to original sources
PubMed1991

Developmental changes of acidic fibroblast growth factor (aFGF) transcription and expression in mouse brain.

In order to increase our knowledge of the in vivo role of acidic fibroblast growth factor (aFGF) in the central nervous system, we have examined aFGF levels during mouse brain development. Using a specific polyclonal antibody raised against aFGF, we measured levels of aFGF-immunoreactive material (IRMaFGF) in extract of total mouse brain taken at different days of development. We found that the level of measurable IRMaFGF remained low and without significant variation during fetal brain development (0.2 ng/mg of extracted proteins). During the first 11 days postnatal (P0 to P11), IRMaFGF increased from 0.5 to 1.5 ng/mg. Between P11 and P14 IRMaFGF levels went up more rapidly, reaching 5 ng/mg. From P30 to adulthood a constant value of 2.5 ng/mg was measured, aFGF content in the different brain extracts was further characterized by its affinity for heparin-Sepharose, its elution at 1 M NaCl from this column and its capacity to induce thymidine incorporation in quiescent fibroblasts. These results were confirmed at the mRNA level. Northern blot analyses of poly A+ mRNA from brains with a specific riboprobe for bovine aFGF, revealed a major 4.5-Kb transcript and a minor 2.7-Kb transcript detectable only in postnatal brains. A similar pattern to that observed for IRMaFGF was seen with these mRNA transcripts, indicating that these aFGFmRNA are translated in the mouse brain. Our results suggest that aFGF may act in the postnatal phases of brain maturation.

Animals↗
PubMed1991

Genomic organization of the human thyroid hormone receptor alpha (c-erbA-1) gene.

The thyroid hormone receptor alpha (THRA or c-erbA-1) gene belongs to a family of genes which encode nuclear receptors for various hydrophobic ligands such as steroids, vitamin D, retinoic acid and thyroid hormones. These receptors are composed of several domains important for hormone-binding, DNA-binding, dimerization and activation of transcription. We show here that the human THRA gene is organized in 10 exons distributed along 27 kbp of genomic DNA on chromosome 17. The position of the introns in human THRA is highly conserved when compared to the chicken gene despite their differing lengths. The N-terminal A/B domain as well as the 5' untranslated region is encoded by two exons. Interestingly, each of the putative zinc fingers of the receptor DNA-binding domain is encoded by one exon and the hormone-binding domain is assembled from three exons. The two last exons of the gene are alternatively spliced to generate two different messenger RNAs. In addition, we confirm that another gene, belonging to the nuclear receptor superfamily, ear-1, overlaps with the 3' region of THRA in an opposite transcriptional orientation.

Amino Acid Sequence↗
PubMed1991

v-myb and v-ets cooperate for the mitogenic stimulation of primary fibroblasts by avian E26 retrovirus.

By using a series of deletion mutants, we have shown that the stimulation of fibroblast growth by E26 requires the cooperation of the two oncogenes, v-myb and v-ets, fused in the nuclear viral product. Of the two DNA-binding domains, only one must be present to promote anchorage-independent growth, whereas that of v-myb is required to allow growth in low serum medium. Furthermore, the v-ets oncogene comprises multifunctional domains.

Animals↗
PubMed1991

An alternatively spliced c-mil/raf mRNA is predominantly expressed in chicken muscular tissues and conserved among vertebrate species.

The chicken c-mil gene produces two mRNA species generated by an alternative splicing mechanism. These two transcripts differ at least by the presence or absence of a 60 nucleotide exon (E7a) localized at the splice junction of c-mil exons 7 and 8. By using RNAase protection assays, we have analysed the pattern of expression of these two mRNA species in several chicken tissues. Here we report that the two c-mil mRNAs are differentially expressed in chicken tissues: the mRNA lacking E7a is detected in all tissues tested, while the mRNA containing E7a is detected only in the skeletal muscle, heart and brain. Sequences homologous to E7a have also been detected in DNAs from quail, mouse and human cells and their sequencing revealed that the alternative E7a is structurally preserved in these species. By PCR analyses performed on RNAs extracted from muscular tissues of these species, we also show that the alternative splicing mechanism described in chicken also occurs in these species.

Amino Acid Sequence↗
PubMed1990

The c-ets proto-oncogenes encode transcription factors that cooperate with c-Fos and c-Jun for transcriptional activation.

Cell transformation by oncogenes leads to changes in gene expression. A key event in this process seems to be activation of the transcription factors AP-1 and PEA 3. Their synergistic activities are required for efficient activation of transcription from different promoters by many different oncogenes, serum growth factors and the tumour promoter TPA. We show here that the products of the ets-1 and -2 proto-oncogenes, whose biological function was previously unknown, are transcription factors that activate transcription through the PEA 3 motif. The p68c-ets-1 protein specifically binds to DNA and contains a transcriptional activation domain. The ets-like gene family therefore seems to encode a new family of transcription factors, apparently unrelated to other transcription factors. The p68c-ets-1 protein cooperates with c-Fos and c-Jun (components of AP-1) for activation of transcription from the oncogene-responsive domain of the polyoma enhancer, indicating that combined activity of all three oncoproteins could be involved in the response of cells to growth stimuli.

Base Sequence↗
PubMed1990

myc products induce the expression of catecholaminergic traits in quail neural crest-derived cells.

The avian myelocytomatosis virus strain MC29 v-myc oncogene transforms a wide panel of avian cells in vitro and either blocks or maintains differentiation, depending on the cell type. In the present work, we have investigated the effect of this oncogene on the differentiation of early embryonic cells, neural crest cells, grown in vitro. We report that the MC29 v-myc gene product induces a strong cellular proliferation of 2-day quail neural crest with the appearance of catecholaminergic traits. Other v-myc as well as the c-myc gene products also trigger this phenotype. Retroviruses carrying some other oncogenes do not elicit this phenotypic expression, although they activate cell multiplication. Thus, our results indicate that myc gene products induce (directly or indirectly) a differentiated phenotype in a subpopulation of neural crest cells.

Animals↗
PubMed1990

The chicken cellular progenitor of the v-ets oncogene, p68c-ets-1, is a nuclear DNA-binding protein not expressed in lymphoid cells of the spleen.

The chicken cellular c-ets-1 locus encodes for two related proteins generated by alternative splicing: the widely expressed p54c-ets-1 protein and the cellular homolog of the v-ets-encoded domain of the E26-transforming protein P135gag-myb-ets, p68c-ets-1 which has been found so far only in the spleen. We have prepared a new site specific antiserum directed against the amino-terminus of p68c-ets-1, which is highly hydrophobic by contrast to the hydrophilic NH2 terminus of p54c-ets-1. This antiserum specifically immunoprecipitated p68c-ets-1 and P135gag-myb-ets only in denaturing and reducing conditions. Despite these biochemical differences at their amino-terminal parts, p68c-ets-1, as p54c-ets-1, is a nuclear protein able to bind to DNA in vitro. Unlike p54c-ets-1 which is expressed at high levels in T- and B-lymphoid cells, p68c-ets-1 is not expressed in lymphoid cells of the spleen. Thus, the two c-ets-1 encoded proteins, although both exhibiting DNA-binding properties in vitro, display differences both in the nature of their specific NH2 termini, and in their level and pattern of expression.

Amino Acid Sequence↗
PubMed1989

Complementary patterns of expression of c-ets 1, c-myb and c-myc in the blood-forming system of the chick embryo.

We have used in situ hybridization to study the spatial and temporal distribution of the transcription of three cellular oncogenes encoding DNA-binding proteins, c-ets 1, c-myb and c-myc during the development of the chick embryo. c-ets 1 mRNA expression appears linked to the mesodermal lineage and is strongly expressed in early endothelia; it subsequently becomes restricted to small vessel endothelia. Hemopoietic cells in extraembryonic blood islands at E2 express c-ets 1, while intraembryonic hemopoietic cells in aortic clusters (E3) and paraaortic foci (E6) express c-myb. c-myc transcripts are detected in cells undergoing hemopoiesis in both these extraembryonic and intraembryonic sites. Outside the blood-forming system, c-myc is transcribed in a large variety of cells; c-ets 1 displays tissue-specific expression in groups of mesodermal cells engaged in morphogenetic processes and appears excluded from all epithelia; finally the expression of c-myb is the most tightly linked to hemopoietic cells. In any case, it is clear that these three oncogenes display complementary expression in endothelial and hemopoietic cells where their patterns are modulated in relationship to multiplication and differentiation.

Animals↗
PubMed1989

A single amino-acid substitution in the DNA-binding domain of the myb oncogene confers a thermolabile phenotype to E26-transformed myeloid cells.

A biologically active provirus of the ts 143 E26 mutant that is temperature-sensitive (ts) for myeloblast transformation was molecularly cloned. The predicted amino-acid sequence of the v-myb-encoded domain of the mutant P135gag-myb-ets protein displayed two single amino-acid changes, one of which was non-conservative when compared to the wild-type E26 v-myb sequence. This mutation, which substitutes a threonine residue (wild-type) for an arginine residue (mutant), is located within the amino-terminal part of v-myb in the DNA-binding domain at a position which is conserved between the c-myb genes of chicken, humans, mice and Drosophila. Introduction of this mutation into the genome of a wild-type E26 virus was sufficient to induce a ts phenotype similar to that obtained with the original ts 143 E26 virus.

Base Sequence↗
PubMed1989

Interstitial deletion of the long arm of chromosome 6.

A de novo interstitial deletion of 6q21 was observed in a male baby with moderate microcephaly, facial dysmorphism, and psychomotor retardation. In situ hybridization with a c-myb probe showed that the gene was conserved on the deleted chromosome.

Chromosome Deletion↗
PubMed1988

Replacement of lys 622 in the ATP binding domain of P100gag-mil abolishes the in vitro autophosphorylation of the protein and the biological properties of the v-mil oncogene of MH2 virus.

Lysine 622 in the ATP-binding domain of P100gag-mil, the translation product of the v-mil oncogene of MH2, has been replaced with methionine using oligonucleotide site-directed mutagenesis. This substitution results in the inactivation of the serine/threonine-specific autophosphorylation of P100gag-mil in vitro, indicating that this activity is an intrinsic property of the viral protein. This substitution also suppresses two of the biological properties of MH2 which have previously been shown to be dependant upon the expression of v-mil, namely, the production of chicken myelomonocytic growth factor (cMGF) by v-myc-transformed chicken macrophages and the sustained proliferation of chicken neuroretina cells. These data strongly suggest that the biological properties of v-mil are mediated by the phosphorylation at serine/threonine residues of key cellular substrates. In contrast to the in vitro situation, both the mutant and wild-type proteins appear to be phosphorylated at the same sites and to the same extent in either transformed fibroblasts or macrophages. This, together with the fact that the sites phosphorylated in vivo and in vitro are essentially different indicate that most of the phosphate associated with P100gag-mil in transformed cells does not result from an obligate autophosphorylation event but from the phosphorylation by as yet uncharacterized cellular kinase(s).

Adenosine Triphosphate↗
PubMed1988

[Cell proliferation and cooperation of v-mil and v-myc oncogenes].

Retroviruses which possess the property to recombine with genetic material from the cell, have cloned and activated some oncogenes and hence are a privileged source for the study of these genes. Cellular oncogene activation can occur following two non mutually exclusive ways: (i) by over-expression of their products; (ii) by modifications of their products through mutations. Retroviruses can combine these two ways of activation leading to the over-expression of a modified product. In this paper, we present results obtained in the study of MH2, a retrovirus containing two oncogenes. We have shown that the two oncogenes of MH2 (v-mil and v-myc) cooperate in vitro to transform neuroretina cells from chicken embryos. These cells which normally do not grow in a defined medium, are induced to proliferate and become transformed upon infection by MH2. Our data enabled us to show that in MH2 v-mil was responsible for the induction of proliferation and v-myc for the transformation of the proliferating cells. Using in vitro constructs we located two regions in the protein encoded by v-mil which are important for its mitogenic property. We have also cloned the cellular counterpart of v-mil and the study of its biological activity on neuroretina cells enabled us to propose a mechanism of activation of the cellular gene by truncation of its 5' part.

Animals↗
PubMed1988

Mapping by in vitro constructs of the P100gag-mil region, accounting for induction of chicken neuroretina cell proliferation.

The v-mil oncogene of the avian retrovirus MH2 is expressed as a fusion protein with viral gag determinants in infected cells. This P100gag-mil protein accounts for the proliferation of chicken embryo neuroretina cells (CNR) induced by MH2 in vitro. We constructed a series of mutants by in-frame deletions in different parts of the gag and mil domains and tested their ability to induce CNR growth. We show that gag sequences, as well as 200-base-pair 5' mil sequences, were not required to induce such a proliferation. However, gag sequences seem to contribute to a full proliferation of growing CNR. In contrast, deletions in the kinase domain abolish this induction. In particular, by deleting only 9 nucleotides localized around the unique SphI site of v-mil, we produced a totally inactive mutant (BalSp). This mutant directs the synthesis of a v-mil protein lacking the dipeptide Tyr-Leu, which is conserved in almost all the members of the large protein kinase family, and a histidine residue highly conserved in Ser-Thr protein kinase members.

Animals↗
PubMed1988

Alternative splicing within the chicken c-ets-1 locus: implications for transduction within the E26 retrovirus of the c-ets proto-oncogene.

Two overlapping c-ets-1 cDNA clones were isolated which contained the alpha and beta genomic sequences homologous to the 5' end of v-ets not detected in the previously described c-ets RNA species or proteins. Nucleotide sequencing demonstrated that these cDNAs corresponded to the splicing of alpha and beta to a common set of 3' exons (a through F) already found in the p54c-ets-1 mRNA. They contained an open reading frame of 1,455 nucleotides which could encode a polypeptide of 485 amino acids with a predicted molecular mass of 53 kilodaltons. However, when expressed in COS-1 cells, the cDNAs directed the synthesis of a protein with an apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 68 kilodaltons, p68c-ets-1, comigrating with a protein expressed at low levels in normal chicken spleen cells. These two proteins were shown to be identical by partial digestion with protease V8. Northern (RNA) blot hybridization analysis with the p68c-ets-1 -specific sequence and RNase protection experiments showed that the corresponding mRNA was expressed in normal chicken spleen and not in normal chicken thymus or in various T lymphoid cell lines. Thus, two closely related proteins, having distinct amino-terminal parts, are generated within the same locus by alternative addition of different 5' exons, alpha and beta or I54, respectively, onto a common set of 3' exons (a to F). Finally, we demonstrate that an aberrant splicing event between a cryptic splice donor site in c-myb exon E6 and the normal splice acceptor site of c-ets-1 exon alpha involved in the genesis of the E26 myb-ets sequence.

Animals↗
PubMed1988

Alternative splicing of RNAs transcribed from the chicken c-mil gene.

Two distinct c-mil-related cDNA clones have been isolated from a chicken embryo cDNA library. Results presented here show that the single chicken c-mil gene is coding for two c-mil mRNA species, different by at least 60 base pairs and generated by an alternative splicing mechanism. These mRNA molecules can be translated into two distinct proteins of 73 and 71 kilodaltons.

Amino Acid Sequence↗
PubMed1988

An invariant asparagine residue belonging to a highly conserved domain in all protein kinases is instrumental in the protein kinase activity of the v-mil gene product.

P100gag-mil, the translation product of the v-mil oncogene of MH2 is a protein kinase specific of serine/threonine residues. We report here that the P100gag-mil encoded by the MH2-Hd isolate displays a considerably reduced kinase activity in vitro. Construction of chimeric viruses and sequencing revealed that the lesion responsible for this reduced activity results from a single point mutation converting an asparagine residue at position 720 in fully active P100gag-mil kinase into serine in the P100gag-mil of MH2-Hd. Since this asparagine residue together with an invariant aspartate residue bracket a highly conserved 6 amino-acid region in all known protein kinases as well as in phosphotransferases of bacterial origin, our results indicate that integrity of this region is essential to enzymatic function and support the notion that it could be directly involved in ATP binding or phosphate transfer from ATP to kinase substrates.

Animals↗
PubMed1988

Cloning and expression of chicken p54c-ets cDNAs: the first p54c-ets coding exon is located into the 40.0 kbp genomic domain unrelated to v-ets.

We have isolated cDNA clones of chicken c-ets mRNA the longest of which, designated pCk E54A, contained approximately 2.0 kb of a c-ets mRNA species. Nucleotide sequencing of this clone revealed a single long open reading frame, extending from the first ATG codon (nucleotide +1) to a TGA termination codon at nucleotide 1324. The predicted translation product contains 441 amino acid residues and its molecular weight is 48 kd. Expression in COS-1 cells of this clone resulted in the synthesis of polypeptides immunologically indistinguishable from the authentic p54c-ets after one-dimensional gel electrophoresis. Comparison of the nucleotide sequence of this cDNA to that of v-ets of avian acute leukemia virus E26 showed that both sequences are almost colinear with the exception of five point mutations but present striking differences in their 5' and 3' parts. 79 nucleotides downstream of the first ATG codon in c-ets cDNA are not found in the 5' part of v-ets where they are replaced by 223 different nucleotides. The 3' parts of v-ets and the coding region of the chicken c-ets cDNAs are also different: the last 13 codons of the cDNA are replaced by 16 different codons in v-ets. Thus our results precisely define the structural differences between the ets encoded domain of E26 viral transforming protein (P135 gag-myb-ets) and the normal cellular protein p54c-ets expressed at high levels in chicken thymocytes and bursal lymphocytes. They also suggest the possibility of alternative splicing of different 5' exons to a common set of 3' exons.

Amino Acid Sequence↗