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Biomedical subjects

M Dion

Publications and source records attributed to M Dion.

At least 19 recordsLinked to original sources
PubMed1991

Repression of the murine interferon alpha 11 gene: identification of negatively acting sequences.

The uninducible murine interferon alpha 11 gene (Mu IFN-alpha 11) shows strong homology with the highly inducible Mu IFN-alpha 4 gene in the promoter region. Negative regulatory sequences located between positions -470 and -145 were characterized in the Mu IFN-alpha 11 promoter. The removal of these sequences leads to virus-inducibility of Mu IFN-alpha 11 while their insertion in Mu IFN-alpha 4 corresponding region significantly reduced the inducibility of Mu IFN-alpha 4 promoter. On the other hand, the virus-responsive element (VRE) of the Mu IFN-alpha 11 differs by a single nucleotide substitution at position -78 from the VRE alpha 4. Constructions carrying either VRE alpha 11 or VRE alpha 4 upstream a heterologous promoter displayed different virus inducibilities. The -78 A/G substitution affects the inducibility by decreasing the affinity of VRE-binding trans-regulators. Our results suggest that the combined effect of the negative regulatory sequences and of the mutation in the VRE alpha 11, completely silences the Mu IFN-alpha 11 gene.

Animals↗
PubMed1991

A new oxygen-regulated operon in Escherichia coli comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (appA)

The Escherichia coli acid phosphatase gene appA is expressed in response to oxygen deprivation and is positively controlled by the product of appR (katF) which encodes a putative new sigma transcription-initiation factor. However, transcription of appA from its nearest promoter (P1) did not account for total pH 2.5 acid phosphatase expression and was not subject to regulation. The cloned region upstream of appA was extended and analyzed by insertions of transposon TnphoA and by fusions with lacZ. It contains two new genes, appC and appB, which both encode extracytoplasmic proteins. appC and appB are expressed from a promoter (P2) lying just upstream of appC. Both genes are regulated by oxygen, as is appA, and by appR gene product exactly as previously shown for appA. Analysis of the nucleotide sequence and of the origins of transcription have confirmed that the P2-appC-appB- (ORFX)-P1-appA region is organized on the chromosome as an operon transcribed clockwise from P2 and that P1 is a minor promoter for appA alone. Genes appC and appB encode proteins of Mr 58,133 and 42,377, respectively, which have the characteristics of integral membrane proteins. The deduced amino acid sequences of appC and appB show 60% and 57% homology, respectively, with subunits I and II of the E. coli cytochrome d oxidase (encoded by genes cydA and cydB). The notion that the AppC and AppB proteins constitute a new cytochrome oxidase or a new oxygen-detoxifying system is supported by the observation of enhanced sensitivity to oxygen of mutants lacking all three genes, cyo (cytochrome o oxidase), cyd (cytochrome d oxidase) and appB, compared to that of cyo cyd double mutants.

Acid Phosphatase↗
PubMed1990

Physical mapping of a temperature-sensitive mutation of human cytomegalovirus by marker rescue.

Physical mapping of a temperature-sensitive (ts) mutation of human cytomegalovirus (HCMV) strain AD-169 was attempted here using cloned HindIII restriction endonuclease fragments and the mutant virus. The DNA-positive mutant tested (HCMV ts 1585) was successfully rescued by viral DNA sequences between 0.277 and 0.303 map units. The product of this gene is apparently a structural protein of molecular weight 40,000. Marker rescue could thus be used to establish the physical location of essential HCMV genes, even if the viral DNA molecule is extremely large and complex.

Chromosome Mapping↗
PubMed1990

Inactivating and mutagenic effects of nitrosoguanide on human cytomegalovirus.

Human cytomegalovirus (HCMV) was exposed for 24 hr to 10-30 micrograms/ml nitrosoguanidine (NG) at different times postinfection. NG treatments performed 24 to 48 hr after infection of human IAFP-1 fibroblasts with HCMV interfered most significantly with the replication of the virus. No correlation was found, however, between the inactivating effect of NG at this particular stage of the replication cycle and the induction of HCMV temperature-sensitive mutants. All the 17 HCMV mutant strains isolated on this occasion showed the same BamHI, Cfo I, Hpa II and Sph I restriction profiles which is indicative of mutations caused by single base changes.

Cytomegalovirus↗
PubMed1990

[Physical mapping of strain AD169 human cytomegalovirus DNA].

The whole human cytomegalovirus strain AD169 genome was cloned into plasmid pAT153 in the form of 25 HindIII fragments. Double and triple digestions of the recombinant plasmids with restriction endonucleases BamHI, BglII, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PaeR7, PstI, SphI and XbaI yielded a detailed restriction map of human cytomegalovirus DNA. Knowing the exact position of numerous restriction sites in the viral DNA molecule, we have been able to examine very closely the heterologous region between the long and the short segments of the human cytomegalovirus genome.

Cloning, Molecular↗
PubMed1989

Expression of the MuIFN alpha 7 gene in Bacillus subtilis using the levansucrase system.

The mouse interferon alpha 7 gene, the signal sequence of which has been removed by oligonucleotide-directed mutagenesis, was introduced into a Bacillus subtilis secretion vector containing the promoter and the signal sequence of the B. subtilis levansucrase gene. Different B. subtilis strains were transformed with the fused levansucrase-interferon gene; their cell extracts and culture supernatants tested for antiviral activity and the IFN alpha 7 protein showed the presence of IFN alpha 7 only in the cell extracts. To promote IFN alpha 7 secretion, constructs were realized in order to restore the alpha helix conformation of the signal sequence of levansucrase and interferon protein junction. Our results suggest that factors other than the structure of the peptide around the cleavage site are involved in the secretion of IFN alpha 7 by B. subtilis.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of a functional murine interferon alpha gene which is not expressed in fibroblasts upon virus induction.

A mouse genomic segment containing three new members of the murine interferon alpha (MuIFN-alpha) gene family was isolated from a fibroblastic cosmid library. A 4 kb EcoRI fragment contained a new MuIFN-alpha gene named MuIFN-alpha 8. The nucleotide sequence of the coding and flanking regions of this gene showed a high level of homology to those of known members of the MuIFN-alpha family. Transient expression of the MuIFN-alpha 8 gene in COS cells and oocyte translation of in vitro transcripts both led to a biologically active protein. The antiviral activity was neutralized by monoclonal and polyclonal MuIFN-alpha antibodies. Although the 5' flanking sequence shows features characteristic of an IFN regulatory region, the MuIFN-alpha 8 gene is not expressed in murine fibroblasts treated with Newcastle disease virus or poly(I).poly(C).

Animals↗
PubMed1988

Recent data on the structure of rabbit milk protein genes and on the mechanism of the hormonal control of their expression.

Mammary explants or isolated mammary cells from rabbit have been cultured in the presence of insulin, prolactin and cortisol alone or in combination. The cellular content in alpha s1-casein, beta-casein and whey acidic protein (WAP) mRNA have been evaluated using the corresponding cDNA as probes. In all cases alpha s1-casein mRNA was the most abundant and WAP mRNA the least abundant mRNA. The three genes showed essentially similar dependency towards hormones. Prolactin stimulated mRNA accumulation and insulin and cortisol amplified this stimulation. The induction by prolactin was rapid whereas stimulation by insulin was slower. Fragments of rabbit genomic DNA inserted in lambda phage and containing alpha s1-casein, and WAP genes have been cloned. The primary sequence around the CAP site of the three genes has been established. A comparison of the sequences located upstream from the CAP site shows several striking homologies with the corresponding genes from cow, rat and guinea-pig. This suggests that these sequences participate in the transcriptional control of the genes by hormones. The mechanism involved in the transduction of the prolactin message to milk protein genes in unknown. Using mammary explants in culture, several classical mechanisms of transduction have been examined. Phorbol ester, phorbol -12, 13-dibutyrate (PdiBu) inhibited prolactin action. However, another tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), did not alter prolactin action. Kinase C inhibitor H7 did not prevent prolactin action and did not overcome the inhibition by PdiBu. Kinase C is therefore not essential for the transduction of the prolactin message to milk protein gene. Neomycin, which inhibits phosphatidylinositol hydrolysis by phosphorylase C, prevented prolactin action, whereas other inhibitors of phosphatidylinositol metabolism remained uneffective. Degradation of phosphatidylinositol is therefore likely not an essential step of prolactin action on milk protein genes. Inhibitors of tyrosine kinase and phosphatase exhibited a poor capacity to modify the prolactin response. Hence, transduction mechanisms using tyrosine kinase activity likely cannot account for prolactin action.

Animals↗
PubMed1987

Relationship between enhanced reactivation and mutagenesis of u.v.-irradiated human cytomegalovirus in normal human cells.

The survival of u.v.-irradiated human cytomegalovirus (HCMV) on u.v.-irradiated human IAFP-1 cells was increased over that on unirradiated cells. Irradiated virus had a higher forward mutation frequency towards temperature sensitivity in irradiated than in unirradiated cells. Enhanced reactivation of u.v.-irradiated HCMV is thus mutagenic in normal human cells. This observation supports the possible induction of an error-prone mode of DNA repair in u.v.-irradiated mammalian cells.

Cell Line↗
PubMed1987

Enhanced reactivation of ultraviolet-irradiated human cytomegalovirus in normal host cells.

Enhanced survival of UV-irradiated human cytomegalovirus (HCMV) is demonstrated in normal human cells exposed to UV light prior to infection. The UV fluence that gave rise to maximum UV reactivation falls in the range of 15 J/m2. A large number of temperature-sensitive HCMV mutants were found under the peak of reactivation. These results confirm the existence of inducible SOS functions in human cells.

Cell Line↗
PubMed1987

Isolation and preliminary characterization of temperature-sensitive mutants of human cytomegalovirus.

Thirteen temperature-sensitive (ts) mutants of human cytomegalovirus (HCMV) have been isolated after mutagenesis with nitrosoguanidine or ultraviolet light. Four mutants defective in viral DNA synthesis at nonpermissive temperature (DNA-) were classified into two separate complementation groups. The other nine HCMV ts mutants, capable of synthesizing viral DNA at the elevated temperature (DNA+), belonged to seven independent complementation groups. The number of essential genes encoded by HCMV is thus raised here by nine.

Cytomegalovirus↗
PubMed1987

Cloning, physical mapping and cross-hybridization of the canine adenovirus types 1 and 2 genomes.

Cloned HindIII fragments of the type 1 and type 2 canine adenovirus (canAd) genomes were mapped with respect to the BamHI, EcoRI, HincII, PaeR7 and PstI restriction endonuclease cleavage sites. Considering the large differences found between the canAd-1 and canAd-2 DNA molecules, in terms of number and location of restriction sites, cross-hybridization experiments were performed. Homologous DNA sequences were located on the canAd-1 and canAd-2 physical maps. Both viruses are genetically related to the extent of 57%. Our results confirm the existence of two distinct Ad species in the dog.

Adenoviridae↗
PubMed1986

Sequence and expression of a novel murine interferon alpha gene--homology with enhancer elements in the regulatory region of the gene.

A murine interferon gene (MuIFN alpha) has been isolated from a cosmid library. The sequence of a 1.2-kb HindIII-PstI fragment revealed a new MuIFN alpha gene which has not yet been described and which was termed MuIFN alpha 7. The coding sequence produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter. A comparison of the MuIFN alpha 7 gene with the known interferon genes in their coding and flanking sequences shows homologies between enhancer elements found in the 5' upstream region of the coding gene. The core element common to all known viral enhancers, GTGG(AAA/TTT)G is repeated four times in the MuIFN alpha 7 5'-flanking region, as in all known MuIFN alpha genes.

Amino Acid Sequence↗
PubMed1986

A simplified procedure for the rapid identification of recombinant pAT153 plasmids in Escherichia coli HB101 cells.

Insertion of foreign DNA into the unique HindIII site of the high copy number plasmid pAT153 reduces but does not completely abolish the resistance of Escherichia coli HB101 cells to tetracycline. Recombinant DNA-containing colonies could then be phenotypically differentiated from non-recombinant ones by their smaller size on nutrient agar plates with ampicillin and tetracycline at a final concentration of 50 and 4 micrograms/ml, respectively. A wide variety of human cytomegalovirus DNA fragments have been found in pAT153 molecules propagated by the ampicillin-resistant tetracycline-sensitive bacteria selected.

Cytomegalovirus↗
PubMed1986

Distinct regions of the human cytomegalovirus genome are responsible for the immortalization and tumorigenicity of animal cells.

Dog embryo kidney cells were efficiently transformed by human cytomegalovirus (HCMV) particles or intact viral DNA. Negative results were obtained after transfection of the canine cells with recombinant plasmids carrying the HCMV Hind III-E subgenomic fragment or with limit Bgl II and Hind III digests of the viral genome. Immortalized dog cells with typical transformation properties appeared, however, after transfection with EcoR I fragments of the HCMV DNA. Distinct regions of the viral genome are probably responsible for the immortalization and the tumorigenicity of mammalian cells.

Cell Line↗
PubMed1984

Increased DNA topoisomerase I activity in aging human cell chromatin.

Chromatin-associated DNA topoisomerase I activity was measured in human diploid fibroblasts during in vitro aging. No difference was detected as a function of cell age in the nicking and the closing activities of the DNA-unwinding enzyme. The capacity of type-I topoisomerase to relax superhelical DNA molecules was, however, increased in aged cells. An age-related increase in nucleoprotein content was also observed.

Aging↗
PubMed1983

Efficient transfection of mammalian cells with viral DNA in optimal culture conditions.

Standard or newly developed DNA transfection procedures were compared for both innocuity and efficiency using simian virus 40 (SV40) DNA and monkey kidney (Vero) cells as an indicator system. Optimal results have been obtained by treating the delicate cell monolayers with a solution of glycerol before infection with DNA-calcium phosphate complexes.

Animals↗