Biomedical subjects
Publications and source records attributed to F Denhez.
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.
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.
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Expression of the P100gag-mil protein of avian retrovirus MH2 in cultured chicken embryo neuroretina cells was previously shown to result in the proliferation of normally quiescent cell populations. We show here that long terminal repeat activation of the carboxy terminus of the c-mil gene is sufficient to induce neuroretina cell proliferation.
We have previously reported that a virus, MH2-PA200, lacking the ability to transform quail embryo cells, could be isolated from wild type (wt) MH2 stocks passaged on chicken neuroretina cells. We report here the molecular cloning and extensive characterization of this MH2-PA200 provirus. Molecularly cloned MH2-PA200 DNA was found to stimulate the growth of neuroretina cells by transfection assays and our results indicate that this recombinant virus was derived from the RAV-1 helper virus, in which v-mil and a small part of v-myc of MH2 were acquired at the expense of helper (delta gag-pol-delta env) sequences. In order to assess the precise boundary between the myc and env genes we determined the nucleotide sequence of the junction fragment and showed that 11 of 13 nucleotides of the env gene were identical to the myc sequence at the recombination point. The nucleotide sequence of the myc-env junction fragment of another similar and independently generated MH2 mutant showed similarly 9 nucleotides of homology between the env and myc sequences at the recombination point that took place at another site, suggesting that a homologous recombination occurred between MH2 and RAV-1 viruses to generate MH2-PA200 and similar mutants.
The genome of the avian retrovirus MH2 contains, in addition to the v-myc oncogene shared with three other avian retroviruses (MC29, CMII and OK-10), a second cell-derived oncogene, v-mil (refs 1-3). Like the three other viruses, which contain only v-myc, MH2 induces mainly liver and kidney carcinomas in fowl and transforms fibroblasts and macrophages in vitro. However, MH2 and MC29 differ in their biological properties when assayed on cultures of chicken embryo neuroretina (NR) cells. Indeed, NR cells, which normally do not multiply in vitro, are induced to proliferate and become transformed upon infection with MH2, whereas infection with MC29 has no apparent effect on these cells. To analyse the functions of the two oncogenes of MH2, we isolated spontaneous and in vitro-constructed mutants of this virus and investigated their effects on NR cell multiplication and transformation. We report here that expression of v-mil is sufficient to induce NR cell proliferation, although it does not result in cell transformation. In addition, viruses expressing only the v-myc oncogene fail to induce any detectable change in NR cells. However, cooperation of the two oncogenes is required to achieve transformation of NR cells by MH2.