Evidence for the direct involvement of DNA replication origin in synthesis of late SV40 RNA.
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Animals↗
Biomedical subjects
W Fiers
Publications and source records attributed to W Fiers.
In vitro cotranslational processing of human pre-interferon beta 1 enhances its biological activity.
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Animals↗
Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes.
A simple and efficient method for direct in vitro synthesis of capped transcripts of cloned eukaryotic genes is described. As an example capped transcripts were made from a plasmid containing the human fibroblast interferon gene cloned under the control of a prokaryotic promoter. These transcripts were translated in vivo in Xenopus laevis oocytes and in vitro in reticulocyte and in wheat germ cell-free protein synthesizing systems.
Animals↗
Complete nucleotide sequence of a human influenza neuraminidase gene of subtype N2 (A/Victoria/3/75).
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Amino Acid Sequence↗
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↗
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↗
Lysis gene expression of RNA phage MS2 depends on a frameshift during translation of the overlapping coat protein gene.
Translation of the lysis cistron of MS2 RNA requires a frameshift of ribosomes reading the upstream coat protein mRNA. The out-of-phase fraction of ribosomes terminates at either one of two nonsense codons just preceding the lysis cistron. Termination is followed by initiation at the start codon of the lysis protein message.
Base Sequence↗
Functional expression of individual plasmid-coded RNA bacteriophage MS2 genes.
The genes of the RNA-containing bacteriophage MS2 were individually inserted into thermoinducible expression plasmids under control of the phage lambda P(L) promoter. Three phage-coded proteins (A-protein, coat protein, and replicase) were expressed at high efficiency. Induced cultures specifically complemented superinfecting amber mutants of phage MS2. Regulatory mechanisms operative during the natural infection cycle of the phage were reproduced by the plasmid expression system.
Journal Article↗
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↗
The sequence specificity of endonucleases CauI and CauII isolated from chloroflexus aurantiacus.
The type II restriction enzymes CauI and CauII, isolated from Chloroflexus aurantiacus, recognize and cleave (at the position indicated by an arrow) the sequences G decreases G A/T CC and CC decreases G/C GG, respectively. These conclusions are supported by the results from restriction site mapping, sequence analysis by partial chemical degradation, end-group analysis after lambda exonuclease treatment and computer-assisted comparison of DNA sequence data.
Bacteria↗
High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter.
Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda. Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500. This 14 500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.
Antibody Specificity↗
Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene.
In the preceding paper (Derom et al., 1981) we described the cloning in bacterial plasmids of the simian virus 40 (SV40) small-t antigen gene under transcriptional control of the bacteriophage lambda pL promoter. Systematic variation of the distance and/or nucleotide sequence between the Shine-Dalgarno ribosome interaction sequence and the small-t translation initiation codon leads to considerable differences in production of small-t by the different plasmids. Secondary structure models derived for the different mRNAs confirm our previous conclusions about the requirement first for an accessible start codon and second for an accessible ribosome interaction site for efficient translation initiation. Secondary structure models for mRNAs from plasmids containing the small-t gene under control of the lac promoter are in agreement with these conclusions.
Antigens, Viral↗
Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes.
By considering the nucleotide sequence of several highly expressed coding regions in bacteriophage MS2 and mRNAs from Escherichia coli, it is possible to deduce some rules which govern the selection of the most appropriate synonymous codons NNU or NNC read by tRNAs having GNN, QNN or INN as anticodon. The rules fit with the general hypothesis that an efficient in-phase translation is facilitated by proper choice of degenerate codewords promoting a codon-anticodon interaction with intermediate strength (optimal energy) over those with very strong or very weak interaction energy. Moreover, codons corresponding to minor tRNAs are clearly avoided in these efficiently expressed genes. These correlations are clearcut in the normal reading frame but not in the corresponding frameshift sequences +1 and +2. We hypothesize that both the optimization of codon-anticodon interaction energy and the adaptation of the population to codon frequency or vice versa in highly expressed mRNAs of E. coli are part of a strategy that optimizes the efficiency of translation. Conversely, codon usage in weakly expressed genes such as repressor genes follows exactly the opposite rules. It may be concluded that, in addition to the need for coding an amino acid sequence, the energetic consideration for codon-anticodon pairing, as well as the adaptation of codons to the tRNA population, may have been important evolutionary constraints on the selection of the optimal nucleotide sequence.
Anticodon↗
Secretory proteins induced in human fibroblasts under conditions used for the production of interferon beta.
Human fibroblast cells treated with a combination of inhibitors of protein and RNA synthesis [cycloheximide and actinomycin D as used to superinduce interferon beta (IFN-beta)] secrete two proteins with molecular masses of 22000 and 27000 kilodaltons (called 22-kDal and 27-kDal) that are precipitable with an antiserum raised against impure IFN-beta but are antigenically distinct from IFN-beta 1. Translation in vitro of mRNA extracted from human fibroblast cells induced for the production of IFN-beta leads to the synthesis of a 26-kDal protein that is structurally closely related to the 22- and 27-kDal proteins. This 26-kDal protein mRNA is relatively abundant and also appears in human fibroblasts induced only with cycloheximide. It has been partially purified by sucrose gradient centrifugation and more extensively by diazobenzyloxymethyl-cellulose hybridization to plasmid DNA from a bacterial cDNA clone. When translated in an in vitro reticulocyte system supplemented with dog pancreas microsomes, the 26-kDal protein and two other intermediates corresponding presumably to its signal-cleaved (19-kDal) and partially glycosylated (24-kDal) forms were observed. Crude, partially purified, and highly purified 26-kDal mRNA failed to program the synthesis of antiviral or ppp(A2'p5')nA synthetase-inducing activity when translated in Xenopus laevis oocytes. Moreover, partially purified 22-kDal and 27-kDal (i.e., the in vivo equivalents of the 26-kDal protein) are also devoid of antiviral or ppp(A2'p5')nA synthetase-inducing activity. Hence, this 26-kDal mRNA, although presumably identical to the human IFN-beta 2 mRNA described by Weissenbach et al. [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, K., Soreq, H., Nir. U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl. Acad. Sci. USA 77, 7152-7156], cannot be considered to be a fibroblast interferon mRNA.
Cells, Cultured↗
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↗
Expression and excretion of human fibroblast beta 1 interferon in monkey cells after transfection with a recombinant SV40 plasmid vector.
We have constructed a eukaryotic expression vector designed to express a gene under late SV40 transcriptional control. From this chimeric plasmic-SV40 vector, virtually all the sequences which code for the major capsid protein VP1 have been deleted and instead, the human fibroblast interferon beta 1 cDNA gene has been inserted. After transfection of monkey cells with this recombinant, substantial quantities of human beta 1 interferon (up to 2 x 10(-4) IU/ml) were excreted in the culture medium. Transfection of nonpermissive mouse L cells or rat cells yielded virtually undetectable quantities of human beta 1 interferon (5 x 10(3) to 10(4) times less than that in monkey cells). The recombinant SV40 vector may serve as a model vehicle for the efficient expression of other eukaryotic genes and might also be used as a direct screening vector for cloning of eukaryotic or prokaryotic cDNA genes.
DNA, Recombinant↗
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↗
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↗