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J Doly

Publications and source records attributed to J Doly.

At least 37 records · Page 2Linked to original sources
PubMed1989

Regulation of the expression of IL-6 in human monocytes.

IL-6 is a cellular regulatory molecule with various cell-dependent functions. We have studied the control of IL-6 expression in human monocytes because they play a key role in the production of this molecule. The effects of adherence and different cytokines including CSF-1, IFN-gamma, IL-1 alpha, and granulocyte-macrophage-CSF were tested on IL-6 expression. IL-6 mRNA was usually not detected in the starting population of PBMC. Adherence induced IL-6 gene expression in monocytes in less than 2 h and subsequently IL-6 secretion. Priming of monocytes by adherence was more efficient for IL-6 overinduction by CSF-1. In contrast, high level induction of IL-6 by IFN-gamma in unfractionated PBMC did not require adherence and in situ hybridization revealed that IL-6 mRNA was present in monocytes but not in lymphocytes. A similar phenomenon was observed for IL-1 alpha and granulocyte-macrophage-CSF. Two cell lines, HL-60 and U937, in which monocytic differentiation occurs after induction by PMA, were subsequently investigated. IL-6 was not constitutively detectable in these two cell lines, whereas PMA treatment induced IL-6 expression. This effect was rapid (30 min) and transitory in HL-60, whereas IL-6 mRNA was still detected after 72 h of induction in U937. Addition of human rIL-6 on U937 and HL-60 cells inhibited their proliferation and enhanced expression of HLA class I Ag.

Cell Adhesion↗
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

Structure and characterization of a murine chromosomal fragment containing the interferon beta gene.

In this paper we report on the cloning and characterization of the murine interferon (IFN) beta gene. We have isolated and sequenced a 2.8 kb genomic fragment containing the murine IFN beta gene flanked by 1.2 kb 5' and 1 kb 3' untranslated regions (1 kb = 10(3) base-pairs). The mRNA cap site has been defined. An extensive analysis of the flanking sequence is provided and points out striking features such as: the presence of A + T-rich motifs characteristic of transiently expressed mRNAs, and homologies to repetitive R-type element flanks and to hormone-responsive elements. Comparison of the MuIFN beta 5' flanking region with those from other species reveals similarities in the sequences required for the regulated expression of such inducible genes. Computer analysis of the 130 base-pairs preceding the cap site has revealed TGAAAG motifs and shows that the presence of such elements and their permutants have biological significance, according to statistical calculations. Thus, the comparison between the mouse promoter reported here and the promoters from other species highlights the region containing the hexanucleotide blocks, which is strongly conserved.

Animals↗
PubMed1988

Purification and carbohydrate structure of natural murine interferon-beta.

Mouse interferon-beta (Mu-INF-beta) induced in C-243 cells with Newcastle disease virus was purified in four steps including ammonium sulfate fractionation. DEAE-cellulose, monoclonal Mu-IFN-beta antibody affinity and Mono-S cation-exchange chromatographies. Specific activity of the purified Mu-IFN-beta ranged over 1.1-1.4 X 10(9) NIH units/mg protein. This preparation was submitted to pronase digestion and gel on Fractogel TSK HW-40. The permethylated and acetylated glycopeptide fraction was analyzed by chemical-ionization (ammonia) mass spectrometry. The major glycopeptide is composed of Gal, Man, GlcNAc and NeuAc with a molar ratio of 2.0:3.6:3.4:0.5. The GLC pattern of methyl derivatives obtained by methanolysis and acetylation of fully methylated glycopeptide identified 2,3,4,6-tetra-O-methylgalactose; 3,4,6-tri-O-methyl-mannose; 2,3,4- and 2,4,6-tri-O-methylgalactose; 2,4,di-O-methyl mannose and 3,6-di-O-methylglucosamine. These results when compared with data on N-glycans suggest the following structure for the carbohydrate moiety of Mu-INF-beta: (formula; see text).

Animals↗
PubMed1987

Isolation and characterization of a cDNA clone encoding testis protamine Z1 from the dog-fish Scylliorhinus caniculus.

A clone containing a 445-bp cDNA insert was isolated from a cDNA library synthesized from dog-fish testes mRNA. The nucleotide sequence was determined and corresponded to a 50-amino-acid protein. The known five-amino-acid N-terminal sequence corresponded exactly to our deduced amino acid sequence. After in vitro transcription of this cDNA using SP6 RNA polymerase, the translated polypeptide comigrated with the Z1 scylliorhinine marker. Analysis of the cDNA 3' flanking region of our Scylliorhinus protamine Z1 revealed an inverted repeat sequence, an ACAA motif and a CAGGAAAGA box known as regulatory signals for transcription termination in histone genes. In addition, sequences homologous to the simian virus (SV 40) and polyoma virus core enhancer elements were identified in the 5' and 3' flanking regions.

Amino Acid Sequence↗
PubMed1986

Gene reorganization during serial divisions of normal human cells.

We have followed during serial divisions of human fibroblasts the presence in chromosomal and extrachromosomal DNA, of two genes that are expressed in fibroblasts, actin and interferon, and of one that is not expressed, globin. The intensity of the blot hybridization of the actin and globin probes with chromosomal DNA diminished during serial divisions of diploid fibroblasts. The interferon gene remained constant throughout the human fibroblast life span. Chromosomal DNA sequences were present in extrachromosomal circular DNA which appeared at the end of the fibroblast life span. The results could explain some functional changes that occur in these cell populations when their division potential declines.

Actins↗
PubMed1986

Nucleotide sequence of a cDNA clone encoding Scylliorhinus caniculus protamine Z2.

A cDNA library was constructed from a protamine-enriched fraction of dogfish (Scylliorhinus caniculus) mRNA. The nucleotide sequence of a 440-bp insert was determined, and its produced protein sequence confirmed its identification as a cysteine-rich protamine Z2 [Martinage, A., Gusse, M., Belaiche, D., Sautiere, P. and Chevaillier, P. (1985) Biochim. Biophys. Acta 831, 172-178]. The frequency of utilization of the different triplets coding for arginine, which represents 30-70% of the total amino acid residues for trout, mouse and dogfish protamines, is discussed. An alternative repetitive sequence of CGC-AGG was found in the N terminus of the protein. Analysis of the 3' flanking region after the mRNA-terminating TAA codon identified an inverted repeat sequence and an ACCA sequence, which may be possible vestiges of a histone-like termination signal.

Animals↗
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↗
PubMed1985

Structure of a murine alpha interferon pseudogene with a repetitive R-type sequence in the 3' flanking region.

A murine alpha interferon pseudogene was identified in a mouse genomic library. The nucleotide sequence revealed several in-phase termination codons within the gene and repetitive oligonucleotides in the flanking regions. The nucleotide sequences and the amino acids of the peptide signal sequences were compared with known human alpha interferon genes and the pseudogene.

Amino Acid Sequence↗
PubMed1985

Structure of mouse interferon-beta cDNA clones: sequence rearrangements in a cloned cDNA.

A fraction enriched in interferon (IFN) mRNA was prepared from mouse C243-3 induced cells and was used for the construction of a cDNA library. Two plasmids were obtained after screening by differential colony hybridization and IFN mRNA hybridization-selection and translation. The nucleotide sequences of the cDNA inserts revealed that both were partial copies of IFN-beta mRNA. The cDNA 861 corresponds to the entire 3' nontranslated region of the mRNA while the cDNA 2939 consists of rearranged translated regions of IFN mRNA. A mechanism for the rearrangement events during cDNA synthesis is proposed. A chromosomal DNA fragment hybridizing to cDNA 2939 was identified by screening a mouse genomic library.

Animals↗
PubMed1981

The primary structure of bovine satellite 1.715.

The primary structure of the 1402 bp repeat unit of bovine satellite 1.715 has been determined using a dimer inserted at the SalI site of plasmid pBR322 and cloned in E. coli. In contrast with bovine satellites 1.706, 1.720b and 1.711a, the 1.715 satellite has a complex sequence with no obvious internal short prototype repeat. The sequence consists however of repeats ranging in length from 6 to 13 nucleotides. In addition, the hexanucleotide, AGATGA, present in the prototype sequences of satellites 1.706, 1.720b and 1.711a, is found in satellite 1.715 in repeats as long as, or longer than, 8 nucleotides, establishing a homology link among those satellites on one hand and satellite 1.715 (and the related satellite 1.711b) on the other. In turn, this suggests a common evolutionary origin. A comparison of the maps for 15 restriction enzymes of cloned and uncloned satellite indicates very little sequence divergence among the repeat units of the latter, most of the differences being due to methylation.

Animals↗
PubMed1981

The chloroplast genome of bleached mutants of Euglena gracilis.

Bleached mutants of Euglena gracilis, traditionally thought to be completely deprived of chloroplast DNA, have been shown to contain a defective chloroplast genome, present at a very low copy number, and preferentially retaining ribosomal RNA genes. We propose to call these mutants phi-, because of their resemblance with the rho- mutants of yeast.

Chloroplasts↗
PubMed1981

Substrate specificity and adenosine triphosphatase activity of the ATP-dependent deoxyribonuclease of Bacillus subtilis.

Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single-stranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The double-stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent Km for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.

Adenosine Triphosphatases↗
PubMed1979

A rapid alkaline extraction procedure for screening recombinant plasmid DNA.

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

Animals↗
PubMed1977

Polyacrylamide gradient electrophoresis for protein purification on the milligram scale.

A preparative-scale electrophoretic technique for protein fractionation and elution on a discontinuous gradient of acrylamide is described, which permits the separation and elution of a pure protein from a mixture containing 4-20 electrophoretically different proteins. The sharpness of the gradient electrophoretic resolution is demonstrated by the separation of proteins consisting of bovine serum albumin polymers and lactate dehydrogenase and enzymes such as acid phosphatase. The compositions of various discontinuous gradients of acrylamide and their application to enzyme purification are discussed. It was found that 60% of the enzyme activity loaded on the gel is recovered after gel fractionation and elution.

Acid Phosphatase↗
PubMed1976

Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis. State of the protein in a mutant devoid of activity.

A prodcedure was developed for the purification of the ATP-dependent deoxyribonuclease of Bacillus subtilis 168. It comprises ammonium sulphate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme has been obtained in a homogeneous state. Its molecular weight was estimated to be 270000 by disc electrophoresis. Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81000, 70000, 62000, 52500 and 42500. These values give 308000 as the molecular weight of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP-requirement for hydrolysis of single-stranded DNA is less strigent. The enzyme also possesses high DNA-dependent ATPase activity. The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits. One subunit is missing (Mr 70000) and is replaced by a smaller polypeptide (Mr 565000). The latter results suggest that the mutant is affected in the genetic locus coding for the 70000-Mr subunit.

Adenosine Triphosphatases↗