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W Fiers

Publications and source records attributed to W Fiers.

At least 379 records · Page 21Linked to original sources
PubMed1981

Evidence for a unique human fibroblast interferon (IFN-beta 1) chromosomal gene, devoid of intervening sequences.

Direct restriction analysis of the human genome, using the Southern transfer technique and hybridization with a human fibroblast interferon (IFN-beta) complementary DNA insert probe, revealed the presence of a single gene; no additional closely related IFN-beta genes could be detected. A lambda-linked human gene library (Lawn et al., Cell, 15, 1157-1174 (1978)) was screened using the cDNA probe. Out of 600,000 recombinant phage examined, one single clone bearing interferon sequences was obtained. Restriction analysis of the relevant region revealed an identical restriction map as obtained for the IFN-beta 1 cDNA clones. No intervening sequences could be detected, either in the coding or the non-coding regions of the gene.

Base Sequence↗
PubMed1981

Initiation of transcription by RNA polymerase II in permeable, SV40-infected or noninfected, CVI cells; evidence for multiple promoters of SV40 late transcription.

CV1 cells were made permeable by treatment with lysolecithin and incubated in a transcription mixture containing ribonucleoside triphosphates including ATP or GTP 32P-labeled either in the alpha or beta position. 5'-terminal cap structures (7mGpgamma pbeta palpha X) on newly synthesized RNA were analyzed by digestion with nuclease P1 or with ribonuclease T2/bacterial alkaline phosphatase. Cap structures obtained after labeling with alpha-32P-GTP show that the 32P is found only adjacent to the 7mG residue (i.e., in the gamma position) and adjacent to the penultimate Gm or G nucleotide (i.e., in the alpha position). Analysis of RNA synthesized in the presence of beta-32P-ATP, however, shows GpppA cap structures which are labeled only in the beta position. In the presence of beta-32-p-GTP, only GpppG structures are labeled; these findings exclude the hypothesis that caps are synthesized from GTP and a monophosphate 5'-terminal RNA molecule. The results imply that the initial transcripts are used for cap formation, which indicates that the large majority (if not all) of capping sites correspond to initiation sites for transcription. In cells infected with wild-types SV40 the distribution of virus-specific caps is similar when labeled either with beta-32P-ATP or with alpha-32P-GTP or with 32p-phosphate. Thus, evidence is presented that heterogeneity of the cap structures in late SV40 is a consequence of independent initiation events and not of processing of a primary transcript followed by capping of the 5' ends generated.

Animals↗
PubMed1981

Nucleotide sequence of the chromosomal gene for human fibroblast (beta 1) interferon and of the flanking regions.

The nucleotide sequence of the human fibroblast (beta 1) interferon chromosomal gene and its flanking regions was determined. These results confirm the absence of intervening sequences in the gene. The presence of some sequences in the upstream flanking region homologous to similar features for other eukaryotic genes was revealed: these include not only the TATAAAT sequence and the consensus sequence (reported by Benoist et al., 1980) but also two additional motifs, one of which is so far present only in inducible genes. Furthermore, a striking similarity between the upstream flanking regions of the human beta 1 and alpha 1 interferon genes is observed.

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↗
PubMed1981

Specific binding of eukaryotic initiation factor 2 to satellite tobacco necrosis virus RNA at a 5'-terminal sequence comprising the ribosome binding site.

The mRNA-binding property of eukaryotic initiation factor 2 (eIF-2) was examined by studying its interaction with satellite tobacco necrosis virus (STNV) RNA carrying a (32)P-labeled 5' end. The RNA molecules bound by limiting amounts of eIF-2 were isolated and digested with pancreatic and T1 RNases. Digestion patterns showed that the labeled STNV RNA preparation offered to eIF-2 was heterogeneous, containing more than 30 different 5' ends; by contrast, the RNA selected by eIF-2 possessed predominantly one 5' end, pApGpUp..., the 5'-terminal sequence of intact STNV RNA. Binding analysis of individual 5'-terminal fragments generated from isolated, intact, STNV RNA by partial digestion with T1 RNase showed that eIF-2 does not bind detectably to the 32-nucleotide fragment ending with the initiation codon AUG or to shorter ones, but it does bind the 44-nucleotide fragment that contains the ribosome binding site. In addition to the structural features localized at the 5' end of STNV RNA, eIF-2 appears to recognize a conformation found only in larger molecules, because intact RNA and large 5-'-terminal fragments are bound preferentially over smaller ones. However, binding of short 5'-terminal STNV RNA fragments to eIF-2 is specific, as judged by competition with STNV and ribosomal RNA. Finally, binding of eIF-2 to intact STNV RNA leads to a conformational change in the RNA that greatly facilitates cleavage by T1 and P1 RNases at sites in the vicinity of the initiation region. These results show that eIF-2 interacts specifically with the 5'-terminal region of STNV RNA that contains the ribosome binding site and causes local unfolding of the RNA structure.

Animals↗
PubMed1981

Phosphorylation patterns of tumour antigens in cells lytically infected or transformed by simian virus 40.

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been analyzed by partial proteolysis peptide mapping and phosphoamino acid analysis of the resulting products. At least four sites were found to be phosphorylated. An amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the proline-rich carboxy-terminal end of the molecule, either at position 701 or at position 708. The mutant dl 1265, which is defective in adenovirus helper function, lacked this phosphorylation site. In addition, the carboxy-terminal part of the molecule contained phosphoserine at a more central position. T-antigen-associated proteins of SV40-transformed cell (nonviral T; 51,000 to 55,000 daltons) also contained multiple phosphorylation sites involving at least two serine residues in mouse antigens and an additional threonine residue in rat, human, and monkey antigens. The latter residue and at least one phosphoserine residue were located near one terminus of the human NVT molecule. We did not find any evidence for phosphorylation of tyrosine residues in any of the multiple species of either large T or nonviral T molecules. Several forms of large T antigens were extracted from both SV40-transformed and SV40-infected permissive and nonpermissive cells, and their phosphorylation patterns were compared. No evidence was found for a different phosphorylation pattern of T antigen in transformed cells.

Animals↗
PubMed1980

Expression of human fibroblast interferon gene in Escherichia coli.

The human fibroblast interferon gene was inserted in a thermoinducible expression plasmid under control of the phage lambda PL promoter. The primary translation products predicted on the basis of the plasmid constructions were hybrid proteins starting with beta-lactamase or phage MS2 polymerase information followed by the total preinterferon. On induction, antiviral activity, whose physico-chemical, immunological and biological characteristics closely corresponded to those of authentic human fibroblast interferon, was synthesized. Processing to a size compatible with mature but unglycosylated authentic product was observed.

Biological Assay↗
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

Secondary structure of mRNA and efficiency of translation initiation.

A series of recombinant plasmids, containing the cro gene of phage lambda, exhibit strikingly different levels of expression depending apparently only on the nucleotide sequence of the untranslated 5' mRNA (Roberts et al., 1979). We postulate that initiation of translation involves interaction between an activated 30S ribosomal subunit and the 5'-terminal region of a messenger RNA already folded in a specific secondary structure. The observed variation in cro synthesis can then adequately be explained by secondary structure models which were derived for the different mRNAs. To maximize expression, it appears necessary that the initiation codon and, although less important, the ribosome interaction site are accessible.

Escherichia coli↗
PubMed1980

Recognition sequence for the restriction endonuclease BglI from Bacillus globigii.

Restriction endonuclease BglI recognizes the DNA sequence (Formula: see text) and cleaves each strand at the site indicated, thus generating 3' protruding ends. The recognition sequence was deduced by correlating mapping data with nucleotide sequence information and the position of cleavage was unambiguously determined by 32P labeling of 5' termini produced by BglI digestion.

Bacillus↗
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↗