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R Devos

Publications and source records attributed to R Devos.

At least 73 records · Page 4Linked to original sources
PubMed1982

The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells.

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.

Amino Acid Sequence↗
PubMed1982

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.

Amino Acid Sequence↗
PubMed1982

Cloning and structure of the human immune interferon-gamma chromosomal gene.

Two clones containing the human immune interferon-gamma (IFN-gamma) chromosomal gene were isolated from a human DNA library present in lambda Charon4A phage. DNA from these clones specified biologically active interferon upon injection into the nuclei of Xenopus laevis oocytes. Analysis of the clones revealed that they were derived from the same chromosomal segment. Restriction fragments that hybridized with 32P-labeled cDNA probes were subcloned into plasmids and the complete sequence of the IFN-gamma gene was determined. Unlike IFNs-alpha and -beta, IFN-gamma does contain introns. Their presence was also revealed by electron microscopy. It is intriguing that the smallest of the three introns is located just in the middle of the Glu-Glu sequence which is conserved among all three forms of interferon at approximately the same position. The promoter region was found to contain a prototype TATA box, many palindromic structures and several repeating sequences and two symmetrical structures. Particularly interesting was the existence of two sequences homologous to those present in the chicken albumin and the human IFN-beta gene promoter region. A sequence GTGTTG common to several other genes was found in the region approximately 10 nucleotides downstream from the polyadenylation site.

Animals↗
PubMed1982

Isolation and characterization of IFN-gamma mRNA derived from mitogen-induced human splenocytes.

Human splenocytes from large cultures (5 1) were stimulated with Staphylococcus enterotoxin A (SEA). After 2.5 d of conditioning, poly A+RNA was isolated from these cultures and its in-vitro translational capacity was evaluated in a wheat germ extract. Injection of the RNA into Xenopus laevis oocytes yielded a low but reproducible amount of IFN activity, which was neutralizable only by an antiserum to gamma interferon (IFN-gamma) and not by antisera to alpha interferon (IFN-alpha) and/or beta interferon (IFN-beta). Formamide sucrose gradient centrifugation analysis indicated that the mRNA which codes for IFN-gamma sediments at around 15 S. The profile, however, suggested a size heterogeneity of IFN-specific mRNA. Electrophoresis of cDNA synthesized on these mRNA fractions indicated that the mRNA coding for gamma IFN has a length of between 900 and 1400 nucleotides.

Animals↗
PubMed1982

The human fibroblast interferon gene(s) and their expression in heterologous cells.

A clone has been identified which contains the genetic information specifying human fibroblast interferon (beta 1) and which was derived from a messenger RNA population. On the basis of the nucleotide sequence the complete amino acid sequence (166 residues) of human fibroblast interferon was deduced. It is preceded by a 21 amino acids long signal sequence. Also, a genomic clone was isolated and characterized in detail; on this basis we can conclude that the interferon beta 1 gene does not contain introns. The nucleotide sequence of the putative promoter region was deduced and compared with other eukaryotic promoters. So far, no evidence has been obtained for other human interferons of the fibroblast type. The interferon beta 1 was expressed in the mature form of Escherichia coli without glycosylation. Fully processed interferon was obtained by transfection of monkey CV1-cells with an SV40-derivative, in which the interferon gene replaced the major structural protein VP1.

Amino Acid Sequence↗
PubMed1981

Complete nucleotide sequence of the nucleoprotein gene from the human influenza strain A/PR/8/34 (HON1).

The complete nucleotide sequence of the influenza A/PR/8/34 nucleoprotein gene was determined after cloning for dsDNA copy in pBR322. The nucleoprotein gene is 1517 nucleotides long of which 1446 nucleotides code for 482 amino acids. The calculated amino acid composition is in good agreement with those published for influenza A nucleoprotein genes. The amino acid sequence of the nucleoprotein contains clusters of basic amino acids and proline, a property shared with other nucleic-acid-associated proteins like Semliki forest virus nucleocapsid protein, VP1 protein of polyoma virus and Simian virus 40, and the core antigen of hepatitis B virus. The described nucleoprotein structure brings the number of sequenced genes of influenza A/PR/8/34 to five out of eight genes.

Base Sequence↗
PubMed1981

Molecular mechanisms of nucleotide-sequence rearrangements in cDNA clones of human fibroblast interferon mRNA.

The characterization and nucleotide sequencing of cDNA clones of human fibroblast interferon (HFIF) mRNA (Derynck et al., 1980) revealed two types of structural inversions :(i) an inverted repeat of a 3'-proximal segment at the 5'end, or (ii) an inverted 5'-terminal segment. Based on the nucleotide sequence of these inverted clones, we have deduced molecular mechanisms to explain how the rearrangements could have arisen. We propose that the usual small 3'-terminal hairpin does not form after the reverse transcription produces the first cDNA strand. Instead, base pairing occurs between the 3'-terminal segment and the more distal region of the first cDNA strand, forming a much larger hairpin loop. Second-strand synthesis followed by either nicking or unfolding of the loop structure and continuation of the polymerase reaction would lead to the observed types of inversions. The 5'-truncated clone, pHFIF-1, whose new 5'-terminal sequence diverges from that of the corresponding region in the mRNA, can also be explained by such a mechanism.

Base Sequence↗
PubMed1980

Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75.

A DNA copy of the gene coding for the influenza A/Aichi/2/68 haemagglutinin protein was cloned in the plasmid pBR322 and the complete nucleotide sequence determined. Comparison of this primary structure and the deduced amino acid sequence with the haemagglutinin gene and protein of strains belonging to the same (H3) subtype and to different subtypes, of both human (H2) and avian (Hav1) origin, documents further at the molecular level the two independent modes of antigenic variation of the virus--drift and shift.

Amino Acid Sequence↗
PubMed1980

Isolation and structure of a human fibroblast interferon gene.

Chimaeric plasmids containing double-stranded cDNA copies of mRNA induced in human fibroblasts by poly I . C were screened by an RNA selection method. A series of clones to which human fibroblast interferon mRNA selectively hybridized was identified. From the nucleotide sequence of the gene, the complete amino acid sequence of human fibroblast interferon was deduced. The protein is 166 amino acids long and is preceded by a 21-amino acid signal sequence.

Amino Acid Sequence↗
PubMed1980

Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA.

The complete sequence of a hemagglutinin (HA) gene of a recent human influenza A strain, A/Victoria/3/75, is 1768 nucleotides long and contains the information for 567 amino acids. It codes for a signal peptide of 16 amino acids, the HA1 chain of the mature hemagglutinin of 329 amino acids, a connecting region between HA1 and HA2 consisting of a single arginine residue and the HA2 portion of 221 amiino acids. The sequence is compared with the hemagglutinin of two members of other subtypes, the human H2 strain A/Jap/305/57 and the avian Hav1 strain A/FPV/Rostock/34, and with one of the same H3 subtype, A/Memphis/3/72. To align the HA1 chain of different major subtypes several deletions/insertions of single amino acids must be invoked, but two more extensive differences are found at both ends, one leading to an extension of the amino terminal sequence of HA1 and the other (four residues) occurring in the region processed away between HA1 and HA2. Comparison of the HA1 of two H3 strains suggests that drift probably depends on single base mutations, some of which change antigenic determinants. The HA2 region, which apparently is not involved in the immune response, is highly conserved even between different subtypes, and single base substitutions account for all the observed diversity. A hydrophobic segment of 24 residues is present in the same position close to the carboxyl terminus of HA2 in both Victoria and FPV, and presumably functions in implantation into the lipid bilayer. The many conserved features not only in HA2 but also in HA1 suggest a rather rigid architecture for the whole hemagglutinin molecule.

Amino Acid Sequence↗
PubMed1978

The 3'-Terminal nucleotide sequence of encephalomyocarditis virus RNA.

Poly(A)-containing encephalomyocarditis virus RNA functions as an excellent template for cDNA synthesis in vitro with an RNA-dependent DNA polymerase in the presence of an oligothymidylate primer. Under appropriate conditions, discrete transcripts of increasing chain length were obtained, suitable for sequence analysis. A limited cDNA fragment of 36 nucleotides, primer (dT)10 included, was synthesized when dGTP was omitted from the reaction mixture and its primary structure was elucidated using direct DNA-sequencing methods. The complement corresponds to the 3' end of encephalomyocarditis RNA. The hexanucleotide (5'-3')(A-A-U-A-A-A) found in this sequence is also present in all 3' non-coding regions of poly(A)-containing eukaryotic mRNAs studied until now, in nearly identical positions relative to the poly(A) tail. The possible biological significance of this structural homology is discussed.

Base Sequence↗