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P Benech

Publications and source records attributed to P Benech.

12 recordsLinked to original sources
PubMed1990

Synergism of type I and type II interferons in stimulating the activity of the same DNA enhancer.

Type I and type II interferons (IFNs) can act synergistically to activate the transcription of the 2-5A synthetase gene. We used in vivo functional assays of sequences from the gene promoter region to determine which DNA segment mediates the gene induction by IFN gamma and the synergistic effect. We found that the type I IFN-inducible enhancer (or IRS) of the 2-5A synthetase gene also confers inducibility by type II IFN to a reporter CAT gene, though the time course and dose response of the induction by the two IFNs are quite different. A clear synergism of the two IFN in stimulating the IRS is observed at low doses of the two IFNs.

Base Sequence↗
PubMed1989

Characterization of human IFN-gamma response using somatic cell hybrids of hematopoietic and nonhematopoietic origin.

A panel of 27 rodent-human somatic cell hybrids composed of cells of hematopoietic (nonadherent cells) and nonhematopoietic origin (adherent cells) was used to identify the chromosomes involved in the biological response to human IFN-gamma (Hu-IFN-gamma). We found that the stimulation of class-I histocompatibility antigen expression correlates with the presence of human chromosomes 6 and 21 in adherent cell hybrids, while human chromosome 6 alone is sufficient in nonadherent hybrids. Scatchard analysis of the binding of radiolabeled Hu-IFN-gamma to nonadherent cell hybrids gave a Kd value similar to that found on human cell lines. Induction of a reporter gene placed under the transcriptional control of the interferon responsive sequence (IRS) in adherent cell hybrids requires both chromosomes 6 and 21. The antiviral protection by Hu-IFN-gamma in adherent cell hybrids was reached at physiological doses (2 units/ml) when human chromosomes 6 and 21 were present, while higher doses of Hu-IFN-gamma (5000 units/ml) were required for hybrids lacking chromosome 21. Thus, we demonstrate that differences exit in the response to Hu-IFN-gamma depending on the origin of the cell type.

Animals↗
PubMed1988

Enhancer-like interferon responsive sequences of the human and murine (2'-5') oligoadenylate synthetase gene promoters.

The human (2'-5') oligo(A) synthetase gene contains two independent cis-acting DNA elements, A and B, which act as transcriptional enhancers. Element A alone is not activated by IFN treatment. Element B alone confers IFN-inducibility to the herpes tk promoter. Two murine (2'-5') oligo(A) synthetase genes were isolated and their promoter sequences show high conservation of element A and B. A synthetic oligonucleotide, containing 16 bp of the human element B, or 14 bp of the homologue murine element B, was linked to a TK-CAT construct. These oligonucleotides were shown to be sufficient to activate the TK promoter in the presence of IFN. When multiple repeats of the interferon-responsive sequence (E-IRS) were cloned in 5' of the TK promoter, the activation ratio was increased. In vitro, specific binding of nuclear protein(s) is observed to the radiolabelled synthetic human E-IRS. This binding is competed by the addition of cold synthetic mouse E-IRS or fragments of genomic DNA containing the E-IRS.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

Four different forms of interferon-induced 2',5'-oligo(A) synthetase identified by immunoblotting in human cells.

Antibodies against synthetic peptides derived from the cDNA sequence of interferon-induced 2',5'-oligo(A) synthetase, and which immunoprecipitate the native enzyme activity, were found to detect multiple enzyme forms in denaturing electrophoretic immunoblots. In some human cell lines, four different interferon-induced proteins of 40, 46, 67, and 100 kDa were found to react with the same peptide antibodies. Each isolated form was shown to have 2',5'-oligo(A) synthetase activity, but the dependence on double-stranded RNA was markedly different for activation of the individual enzymes. The four enzyme forms also differ in their intracellular localization, on microsomes (100 kDa), in nuclei (67, 46, 40 kDa), and on membrane structures (67 kDa). Plasma membranes from interferon-treated Daudi lymphoblastoid cells are highly enriched in the 67-kDa 2',5'-oligo(A) synthetase form. The 2',5'-oligo(A) synthetase activity induced by interferons in human cells appears, therefore, as a complex multienzyme system.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

Interferon-responsive regulatory elements in the promoter of the human 2',5'-oligo(A) synthetase gene.

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

3' end structure of the human (2'-5') oligo A synthetase gene: prediction of two distinct proteins with cell type-specific expression.

Analysis of the sizes of the (2'-5') oligo A synthetase polyadenylated transcripts in various human cell lines by hybridization with a cDNA probe revealed that the pattern of the transcripts was cell-type specific. In order to investigate whether the 1.6 Kb, 1.8 Kb and 3.6 Kb mRNA, which are predominantly expressed in different cell-lines, encode different proteins or could differ in the size of their 3' or 5' untranslated sequences, the corresponding gene was isolated. On Southern blots of human genomic RNA, the cDNA probe hybridize to a 3.1 Kb EcoRI fragment. Three overlapping genomic clones were isolated which contain the same 3.1 Kb EcoRI fragment. Transcriptional mapping of the 3' end of the gene by Northern blots, hybridization translation experiments and sequencing showed that the 1.6 and 1.8 Kb mRNA are produced by a differential processing at the 3' end of the primary transcript of the same gene. The polyadenylation site of the 1.6 Kb mRNA is located 1.9 Kb upstream to the one of the 1.8 Kb mRNA. To produce the latter transcript, a portion of the last exon of the 1.6 Kb mRNA (including a segment coding for a very hydrophobic sequence of 18 amino acids and 3' untranslated sequence) is spliced out. The two mRNAs thus encode two different proteins which could have different affinity for cellular elements. The 3.6 Kb transcript has the same polyadenylation site as the 1.8 Kb mRNA, but contains additional unspliced 3' intron sequences.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

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.

2',5'-Oligoadenylate Synthetase↗
PubMed1983

Interferon-induced 56,000 Mr protein and its mRNA in human cells: molecular cloning and partial sequence of the cDNA.

Treatment of responsive cells by interferons (IFNs) induces within a few hours a rise in the concentration of several proteins and mRNAs. In order to characterize these IFN-induced mRNA species, we have cloned in E. coli the cDNA made from a 17-18S poly(A)+ RNA of human fibroblastoid cells (SV80) treated with IFN-beta. We describe here a pBR322 recombinant plasmid (C56) which contains a 400 bp cDNA insert corresponding to a 18S mRNA species newly induced by IFN. The C56 mRNA codes for a 56,000 dalton protein easily detectable by hybridization-translation experiments. The sequence of 66 of the carboxy-terminal amino-acids of the protein can be deduced from the cDNA sequence. IFNs-alpha, beta or gamma are able to activate the expression of this gene in human fibroblasts as well as lymphoblastoid cells. The mRNA is not detectable without IFN; it reaches maximum levels (0.1% of the total poly(A)+ RNA) within 4-8 hrs and decreases after 16 hrs.

Amino Acid Sequence↗
PubMed1983

Molecular cloning and sequence of partial cDNA for interferon-induced (2'-5')oligo(A) synthetase mRNA from human cells.

By using a translation assay in oocytes, a 17S RNA fraction coding for the interferon-induced (2'-5')oligo(A) synthetase was purified from human cells. A cDNA library was prepared by cloning in Escherichia coli plasmid pBR322 and screened by positive hybridization-translation in oocytes. A cDNA clone corresponding to the (2'-5')oligo(A) synthetase mRNA was identified. In SV80 cells, this E cDNA recognizes three RNAs of 1.65, 1.85, and 3.6 kilobases, which are present only after interferon treatment of the cells. In Namalva cells, mainly one RNA of 1.8 kilobases is seen.

2',5'-Oligoadenylate Synthetase↗
PubMedDate not supplied

Constitutive expression of (2'-5') oligo A synthetase confers resistance to picornavirus infection.

Study of the mechanisms by which interferon (IFN) treatment of cells induces resistance to virus infections has been complicated by the multiple biochemical changes induced. Over 20 proteins are increased by IFN, including the double-stranded (ds) RNA-activated protein kinase, (2'-5') oligo A synthetase, surface proteins such as the major histocompatibility complex (MHC) proteins, and various proteins with unknown functions. The availability of cloned complementary DNAs for several IFN-induced proteins now allows us to probe their roles in IFN action. For instance, the murine Mx protein has been shown to confer resistance, to influenza virus. We studied chinese hamster ovary (CHO) cell clones expressing high constitutive levels of (2'-5') A synthetase as a result of transfection with the cDNA encoding the enzyme form which has a relative molecular mass (Mr) of 40K. Elevated enzyme correlates directly with resistance to infection by a picornavirus such as Mengo, but does not make the cells resistant to vesicular stomatitis virus (VSV).

2',5'-Oligoadenylate Synthetase↗