Yves Mory

Structure of two forms of the interferon-induced (2'-5') oligo A synthetase of human cells based on cDNAs and gene sequences.

The (2′‐5′) oligo A synthetase E, one of the translational inhibitory enzymes whose synthesis is strongly induced by all interferons (IFNs), is shown to be encoded in human cells by a 13.5‐kb gene. By a cell‐specific differential splicing, between the seventh and an additional eighth exon of this gene, two active E mRNAs of 1.6 and 1.8 kb are produced, along with several longer transcripts. cDNA clones for the two mRNAs were obtained and their sequences indicate that the human (2′‐5′) oligo A synthetase gene codes for two forms of the enzyme of mol. wt. 41 000 and 46 000, which differ only by their C‐terminal ends. The product of the 1.6‐kb RNA (E16) has a very hydrophobic C terminus, which is replaced by a longer acidic C‐terminal sequence in the 1.8‐kb RNA product (E18). The transcriptional start site of the gene was identified and 200 bp of the 5′ flanking region were sequenced. A strong homology was found between this region of the IFN‐activated (2′‐5′) oligo A synthetase gene and the corresponding region of the human fibroblast IFN‐beta 1 gene, whose transcription is also stimulated by IFN priming. The gene has two polyadenylation sites which share a common undecanucleotide, but are used in a cell‐specific manner to give rise to the 1.6‐ and 1.8‐kb mRNAs.

Inducible expression of the human interferon beta 1 gene linked to a bovine papilloma virus DNA vector and maintained extrachromosomally in mouse cells.

A 1.6-kilobase DNA segment of the genomic human interferon beta 1 (IF-beta 1) gene was inserted into each of two possible orientations at the single HindIII site of a recombinant plasmid pBPV69T, consisting of the 69% transforming region of the bovine papilloma virus type 1 (BPV-1) and a modified SalI-SalI fragment of plasmid pBR322. After cleavage of the pBR322 sequences from this recombinant, BPV69T-IF-beta 1 hybrid DNAs were transfected onto C127 mouse cells by the standard calcium precipitation technique. Mouse cells transformed by this hybrid DNA produced low levels of human IF-beta 1 constitutively and responded to induction with either inactivated Newcastle disease virus or polyriboinosinic acid-polyribocytidylic acid. The BPV69T-IF-beta 1 hybrid DNA was nonintegrated in the transformed mouse cells but had acquired DNA sequences as a result of the transfection. Accurate transcripts of the IF-beta 1 mRNA were detected in cells only after induction. When the IF-beta 1 gene was oriented in the plasmid in the same direction of transcription as the BPV-1 genome, transcription was promoted from within the BPV-1 sequences. These results indicate that the regulatory sequences responsible for the inducible expression of the human IF-beta 1 gene are present in the 1.6-kilobase genomic segment and that these sequences can function in a free extrachromosomal state linked to BPV-1 sequences.

Family of human alpha-interferon-like sequences.

An interferon-alpha-like sequence was isolated from a human genomic library by hybridization with a 15-base oligonucleotide. The sequence also showed homology to alpha-interferon and was most closely related to the leukocyte interferon-M gene fragment. The original isolate cross-hybridized to a family of sequences, 10 of which were isolated as clones. Some of these sequences were located within a few kilobases of alpha-interferon genes, consistent with our assignment of several members of the family to human chromosome 9 which also has the beta 1- and alpha-interferon genes.

Sequences involved in the regulated expression of the human interferon-beta1 gene in recombinant SV40 DNA vectors replicating in monkey cells.

The human genomic EcoRI fragment of 1.83 kb containing the interferon (IFN) gene IFN‐beta1 with 285 nucleotides of 5′‐flanking sequences was transfected into monkey kidney CV‐1 cells as part of an SV40‐pML2 vector. Induction of the monkey cells to produce IFN led to a rapid accumulation of IFN‐beta1 RNA whose 5′ ends were identical to the IFN‐beta1 mRNA of human fibroblasts. This induction occurred with all recombinants tested. Expression from the SV40 late promoter was also seen in non‐induced cells. We conclude that the regulation of the IFN‐beta1 gene is retained in the replicating episomal SV40 vectors with high copy number, even when the gene is being transcribed from an external promoter. When the 5′‐flanking sequences were deleted to leave only 40 bp before the presumed cap site of the IFN‐beta1 gene, inducible formation of IFN‐RNA with authentic 5′ ends could still be demonstrated. However, inducibility and expression depended on the position of the deleted IFN‐beta1 gene in the vector. We conclude that the sequences around the TATAA box and cap site on the IFN gene are involved in the regulation of its expression. Regulated short‐term expression of the human IFN‐beta1 gene in SV40 vectors provides a defined system in which the structures required to maintain the regulation and the influence of known external transcription signals can be examined.

Analysis of mouse embryonic gene library for the frequency of single and multiple copy genes

A gene library was constructed from embryonic mouse DNA by ligating DNA fragments generated by partialEco RI digestion with Charon 4A vector andin vitro packaging. A special consideration was given to randomization of target DNA. The general applicability of a gene library prepared in this manner was assessed through cloning a variety of genes of known reiteration frequency in the mouse genome. The survey included a single copy gene — C region of the immunoglobulin heavy chain, and genes that appear in more than one copy — V region of the immunoglobulin light chain genes and the endogenous retrovirus related genes. In all cases tested the frequency of clone isolation was in good agreement with the expected incidence based on the number of genome equivalents screened and the reiteration frequency of that particular gene. Moreover, we found no preference with regard to the clonability of genes contained in fragments of a wide-size range.

Biological Activities of Human Interferon-β2 Produced by cDNA Expression in Hamster Cells and Possible Autocrine Functions of this Cytokine-Induced IFN

Human fibroblasts induced by poly (rI)(rC) and sequential cycloheximide/Actinomycin D treatment produce, in addition to IFN-β1 and its 0.9 kb mRNA, another mRNA of 1.3 kb encoding IFN activity neutralized by anti-IFN-β antibodies and hence designated IFN-β 2 (1,2). An IFN-β2 cDNA was cloned (1) and used to screen a human genomic library from which two intron-containing genes IFA-2 (IFN-β2a) and IFA-11 (IFN-β2b) were identified (3). The IFN-β2a gene is 4.8 kb long, and was mapped on human chromosome 7 (4). Both genomic clones have been expressed in rodent cells and produced human IFN antiviral activities (5). The recombinant IFN-β2 induces (2′–5′) oligo A synthetase mRNA and HLA mRNAs in the presence of cycloheximide (3,6) and has antiviral activity on mouse-human hybrid cells containing human chromosome 21 (but not 9), excluding the possibility that IFN-32 acts through IFN-β1 induction (3,6). By immunocompetition with the in vitro translation product (23–26 Kd) of IFN-β2 mRNA, the native form of IFN-β2 secreted by human cells, was identified as a 21–22 Kd glycoprotein (3). Native IFN-β2 can be separated from IFN-β1 because it is not retained on Blue-Sepharose and elutes from DEAE-cellulose pH 7.4 at lower salt (150 mM NaCl) than IFN-β1.

The Use of Ion Exchange and Reversed-Phase HPLC for the Isolation and Characterization of Interferon Gamma

Publisher Summary Human interferon-γ (IFN-γ) is a lymphokine that is produced by peripheral blood mononuclear cells upon stimulation with mitogens, such as phytohemagglutinin (PHA). IFN-γ consists of two molecular subtypes having apparent molecular weights of 26,000 and 21,000 as determined by sodium codicil sulfate -polyacrylamide gel electrophoresis (SDS-PAGE). On gel filtration, a single peak having an apparent molecular weight of 45,000 is obtained. Analysis of IFN-γ cDNA revealed the presence of only a single gene, coding for a polypeptide of 166 amino acid residues, which includes a leader peptide of about 20 amino acid residues. IFN-γ is a glycoprotein having two glycosylation sites and lacking disulfide bridges. Unlike IFN-α and IFN-β, it is labile at low pH. The present study characterizes the structure of IFN-γ.

Interferon-type and Other Activities of IFN-ß-2/BSF-2/HSF

The multiple activities of IFN-β-2 (BSF-2, IL-6, 26 Kd, HGF, HSF) suggest that this multifunctional cytokine plays a role in the various aspects of inflammation resulting from pathogenic infections.

Monoclonal Antibodies for Purification and RIA of Natural and Recombinant Human Interferon-α, -β And-γ

Abstract Monoclonal antibodies were prepared against three major groups of human interferons. A novel screening procedure was developed and used for the selection of specific hybridomas. Immunoadsorbents prepared from these antibodies were used for purification to homogeneity of human interferons from various sources. Low pH was used for recovery of interferon-α and -β, high pH was used for interferon-γ. Both recombinant IFN-α from E. coli and several types of cellular interferon-α were purified to homogeneity as determined by SDS-polyacrylamide gel electrophoresis and by the specific activity. Interferon-β produced by either normal fibroblasts or by recombinant CHO cells was purified to homogeneity by immunoaffinity chromatography. Similarly, interferon-γ produced by either peripheral blood mononuclear cells or by recombinant CHO cells was purified to homogeneity in one step. Double antibody solid phase RIAs for IFN-α, -β and -γ were developed.