André Renard

Isolation and characterization of the human prolactin gene

Prolactin (PRL) and growth hormone (GH) genes derive from a common ancestor and still share some sequence homologies. Their expression in the pituitary gland is regulated in opposite directions by most of the many hormones acting on them. This provides an interesting system to study sequences involved in gene expression. Using a human PRL cDNA clone as a probe, we screened a human genomic DNA library in lambda phage and isolated a single recombinant comprising the whole hPRL gene. It was characterized by restriction endonuclease mapping and cDNA hybridization, by DNA heteroduplex analysis and by nucleotide sequencing. The hPRL gene is present as a single copy per haploid genome, is approximately 10 kb long and contains four introns, three of which interrupt the coding sequence at the same locations as in the known GH and PRL genes. The origin of transcription was determined by S1 mapping on prolactinoma mRNAs. The search for direct and inverted repeats, as well as dyad symmetries was carried out in the 900‐bp sequenced in the 5′‐flanking region. Sequence homologies between hPRL, hGH and rPRL were derived from computer drawn matrices for these upstream regions.

Expression in Escherichia coli of two mutated genes encoding the cholera toxin B subunit

To allow subsequent genetically mediated fusion of foreign antigens to cholera toxin B subunit (CTB), two mutated CTB encoding genes (ctxB) were constructed and overexpressed in Escherichia coli. The signal peptide coding sequence was deleted and restriction sites were created at both ends of the modified sequence. Both synthesized CTBs contain additional amino acid(s) at the N terminus (one and three). They were purified as insoluble products and refolded into the natural pentameric CTB structure by a denaturation-renaturation cycle. After renaturation, both recombinant proteins recovered CTB antigenicity and the ability to bind to GM1 gangliosides, as shown by in vitro analysis. Preliminary data indicated that both properties were unaltered by fusion of a foreign peptide to the mutated CTBs.

Expression of the bovine viral diarrhoea virus Osloss p80 protein : its use as ELISA antigen for cattle serum antibody detection

The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoter-based vector for expression in Escherichia coli. Bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. Rabbit antisera were raised against this p80 recombinant antigen and assayed for the immunoprecipitation of either p120 or p80 protein from cytopathic or non-cytopathic BVDV biotype-infected bovine cells. The p80 gene sequence was also integrated into a baculovirus genome for its expression in Spodoptera frugiperda insect cells. The recombinant proteins isolated from bacteria or insect cells showed distinct antigenic properties when analysed by ELISA. Their ability to detect anti-BVDV specific antibodies was examined in a monoclonal antibody-based competitive ELISA performed on a series of field cattle sera. This comparative assay revealed the superiority of the insect cell-mediated expression to mimic the natural BVDV antigen produced by cell culture. The baculovirus/insect cell recombinant antigen gave the highest correlation between the ELISA-detected antibodies and the corresponding virus neutralization data.

A chromatin factor in rat liver which destroys O6‐ethylguanine in DNA

Adapted Escherichia coli cells (i.e. pretreated with a low concentration of N-methyl-N’nitro-N-nitrosoguanidine) contain a factor able to promote the disappearance of U6-methylguanine from DNA [ 1,2]. The 06-methylguanine is not released as a free base and the transformation product remains in DNA [2]; the factor seems to be active at 0°C and to disappear during the reaction [ 11. Renard et al. [3] observed the disappearance of @-ethylguanine from the DNA of isolated rat liver nuclei treated in vitro with ethylnitrosourea and Pegg [4] has found, in the total proteins of a rat liver extract, an activity which induces the disappearance of @-methylguanine from DNA. As in E. co/i, the glycosylic bond is not hydrolyzed to release free @-methylguanine. In this communication, we show that, in rat liver, chromatin has the highest concentration of the factor capable to decrease the @‘-ethylguanine content of an added ethylated DNA. Some properties of the chromatin factor are also presented.

Stabilization of T7-promoter-based pARHS expression vectors using the parB locus

We describe a modification of the pAR3040 vector which results in its efficient stabilization during cell division. The parB locus of the plasmid R1 was introduced into the plasmid, pAR3040, to construct the pARHS vectors. These vectors are stable for at least 60 cell generations, even in the absence of selection by an antibiotic present in the culture media, both with or without IPTG induction.

A common chromatin factor involved in the repair of O6-methylguanine and O6-ethylguanine lesions in DNA

Repair of DNA containing 06-methylguanine or 06-ethylguanine by rat liver total proteins was observed in [ 1,2]. We have shown that the factor responsible for the disappearance of 06-ethylguanine is mostly located in chromatin [3] and that the ethyl group is transferred onto 2 cysteine residues of acceptor proteins [4]. Using a factor partially purified from mouse liver supernatant repair of DNA containing 06-methylguanine and transfer of the methyl group onto cysteine residues of proteins was shown in [S]. This work shows that chromatin roteins from rat liver contain factors acting on f? 0 -methylguanine as well as 06-ethylguanine in DNA. Competition experiments indicate that a limiting component is common to both repair systems.

Identification and production of pestivirus proteins for diagnostic and vaccination purposes

Using a panel of monoclonal antibodies (MAbs) previously characterized by seroneutralization, immunofluorescence and radioimmunoprecipitation, we have identified Pestivirus proteins useful for diagnostic purposes from the cytopathic Osloss isolate of bovine viral diarrhea virus (BVDV). Proteins that should be useful for vaccination have also been analysed. Cell-free translation of RNA from glycoprotein-coding cDNA fragments produced, when synthesized in the presence of canine pancreatic microsomes, two glycosylated proteins that were independently recognized and immunoprecipitated by two distinct classes of neutralizing MAbs. A similar in vitro procedure was carried out on nonstructural protein-coding sequences and allowed to identify a viral translation product that specifically reacted with MAbs directed against the 80 kDA protein of a number of Pestivirus strains. Its positioning within the polyprotein encoded by the viral genome was refined by epitope scanning using synthetic hexameric peptides. This viral antigen was further expressed in E. coli, produced as inclusion bodies and used successfully as an ELISA antigen in both competitive and indirect assays for the detection of BVD antibodies in cattle sera.

Kinetic analysis of O6-ethylguanine disappearance from DNA catalyzed by the chromatin factor of rat liver.

Rat liver chromatin contains a factor which induces the disappearance of O’-ethylguanine from DNA alkylated with ethylnitrosourea f 1,2]. The transformation product has not yet been isolated nor, of course, identified. We noticed that, in contrast to the situation in Escherichia coli [3,4], this factor is present constitutively in chromatin and that it seems to be an enzyme rather than a stoichiometric reagent: it is inactive at 0°C and, when there is an excess of substrate, it is still working after 120 min at 37’C. Here, we give additions details on the chromatin repair factor showing that it behaves as an enzyme which is competitively inhibited by the reaction product.

A "zinc finger-like" domain in the 54KDA protein of several pestiviruses.

We sequenced cDNAs, amplified by the Polymerase Chain Reaction (PCR) which correspond to the carboxy-terminal portion of the 54 KDa protein of various cytopathic (cp) or non-cytopathic (ncp) pestiviral strains. Except for the previously described insertions in two cp strains of the Bovine Viral Diarrhea virus (BVDV), we did not find comparable insertions in this gene. The predicted amino acid sequences of this 54 KDa protein portion contain a conserved cysteine-rich stretch remarkably similar to a “zinc finger”-type binding domain found in many gene-regulatory proteins. Thus, this protein may be involved in the binding to nucleic acids. As the 54 KDa protein is released from its 125 KDa precursor only in the cp strains, we propose the cytopathology may be a consequence of this event.

Repair of O6-alkylguanine lesions in DNA by chromatin enzymes.

Disappearance of 7-ethylguanine and O6-ethylguanine lesions from nuclear DNA is observed on incubation of isolated rat liver nuclei previously treated with ethylnitrosourea. Free 7-ethylguanine but no free O6-ethylguanine is released in the medium. Incubation of methylated or ethylated DNA with chromatin proteins leads to the disappearance of O6-methylguanine or O6-ethylguanine from DNA; the methyl group or the ethyl group is transferred to proteins where it is accepted by cystein residues. Repair of O6-methylguanine and repair of O6-ethylguanine lesions in DNA consume a common reactant which is likely the acceptor protein. The repair does not seem however to be a simple bimolecular reaction between the O6-alkylguanine and the cystein of the acceptor protein.