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C Weissmann

Publications and source records attributed to C Weissmann.

At least 91 records · Page 5Linked to original sources
PubMed1990

A novel, quantitative bioassay for type I interferon using a recombinant indicator cell line.

We describe a specific and quantitative novel assay for biologically active human type I interferon (IFN), the MxR assay. It is based on a Vero cell line containing multiple copies of a hybrid gene consisting of the murine Mx promoter, which is responsive to type I IFN, linked to the human growth hormone (hGH) transcription unit. Exposure of this cell line to IFN-alpha or -beta for 12-48 hours results in the production of hGH that is measured by a commercially available radio-immune assay. The response to IFN-alpha is dose-dependent between 3 and 1000 units/ml. There is no response to TNF, IL-1 and a number of other cytokines and growth factors, and only a negligible response to IFN-gamma.

Animals↗
PubMed1988

Search for a scrapie-specific nucleic acid: a progress report.

Scrapie agent contains a proteinaceous component as well as an 'informational' molecule (suggested by the existence of distinct strains of scrapie). These operationally defined entities may be the same molecule, an infectious protein, or distinct, in which case a nucleic acid might encode the genetic information. Purification of scrapie agent enriched a protein, PrPSc, by virtue of its relative protease resistance. There is only a single PrP gene and the primary translation product of PrP mRNA is the same in normal and scrapie-infected brain; therefore the normal PrPC and the protease-resistant isoform, PrPSc, found in scrapie, probably result from different post-translational events. To search for scrapie-specific nucleic acid, globin RNA made in vitro was added to highly purified infectious preparations at a ratio of 10(3) molecules per infectious unit, nucleic acids were isolated and denatured, and cDNA synthesized using random oligonucleotide primers. Clones containing globin-related sequences were identified by in situ hybridization. 150 plaques not hybridizing to the globin probe were isolated. Inserts larger than 50 base pairs were analysed. By hybridization to a globin probe at reduced stringency all but four clones were found to contain small globin related inserts; two of these hybridized to hamster repetitive sequences as shown by Southern blot analysis. The other clones not related to hamster nucleic acids may be derived from unknown sources of contamination or from scrapie-specific nucleic acids.

Animals↗
PubMed1988

Organization of the murine Mx gene and characterization of its interferon- and virus-inducible promoter.

Specific resistance of Mx+ mice to influenza virus is due to the interferon (IFN)-induced protein Mx. The Mx gene consists of 14 exons that are spread over at least 55 kilobase pairs of DNA. Surprisingly, the Mx gene promoter is induced as efficiently by Newcastle disease virus as it is by IFN. The 5' boundary of the region required for maximal induction by both IFN and Newcastle disease virus is located about 140 base pairs upstream of the cap site. This region contains five elements of the type GAAANN, which occurs in all IFN- and virus-inducible promoters. The consensus sequence purine-GAAAN(N/-)GAAA(C/G)-pyrimidine is found in all IFN-inducible promoters.

Amino Acid Sequence↗
PubMed1987

Reversible silencing of enhancers by sequences derived from the human IFN-alpha promoter.

The virus-responsive element of the IFN-alpha 1 promoter, VRE(IFN alpha), comprises two imperfect 19 bp repeats, repA and repB. VRE(IFN alpha), tetrameric repA, and tetrameric GAAAGT (a subsequence of repB) or tetrameric AAGTGA conferred inducibility on a reporter gene when placed upstream of a complete or truncated promoter. Induced transcription was weak with a minimal promoter (TATA box only), but was strongly stimulated by the SV40 enhancer placed immediately upstream of the inducible element. Surprisingly, under noninduced conditions, tetrameric repA, GAAAGT, and AAGTGA (but not VRE(IFN alpha)) completely silenced enhancement of constitutive transcription by the SV40 72 bp repeat when interposed between the latter and the TATA box; silencing was fully abrogated by induction.

Animals↗
PubMed1987

5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU.

We have generated all possible single point mutations of the invariant 5' GT of the large beta-globin intron and determined their effect on splicing in vitro. None of the mutants prevented cleavage in the 5' splice region, but many reduced or abolished exon joining. The mutations GT----TT and GT----CT resulted in a shift of the 5' cleavage site on nucleotide upstream; in the case of the mutation GT----TT, this shift was reverted by a second site mutation within the 5' splice region. Our results suggest that the 5' cleavage site is determined not by the conserved GU sequence but by the 5' splice region as a whole, most probably via base-pairing to the 5' end of the U1 snRNA.

Animals↗
PubMed1987

Induction of mRNA for a serine protease and a beta-thromboglobulin-like protein in mitogen-stimulated human leukocytes.

Two cDNA clones corresponding to genes that are induced at least 10-fold in peripheral human blood leukocytes by staphylococcal enterotoxin A were isolated and sequenced. Clone 1-3E encodes a 247-residue protein that comprises a putative signal sequence, and resembles a serine protease; the cognate mRNA is expressed in T lymphocyte clones but in none of the other human cell lines tested. The deduced protein sequence is most closely related (68% homology) to that of the postulated protease CCPI from activated murine cytotoxic T lymphocytes and to that of rat mast cell protease II (47% homology). The other cDNA, 3-10C, encodes a protein of 99 residues that resembles human beta-thromboglobulin (42% homology); the cognate mRNA was also found in SEA-stimulated U937 cells, a histiocytic lymphoma-derived cell line.

Amino Acid Sequence↗
PubMed1987

Single amino acid changes that render human IFN-alpha 2 biologically active on mouse cells.

Human IFN-alpha 1 and IFN-alpha 2 differ in 28 of 166 amino acids and show very different specific antiviral activities on human and murine cells. We have identified, by hybrid scanning and site-directed mutagenesis, three residues in IFN-alpha 2, in positions 121, 125 and 132 which, when replaced individually or jointly by their IFN-alpha 1 counterparts, modify its activity on mouse cells by up to 400-fold. We argue that these residues are involved in direct contacts with the mouse interferon receptor.

Amino Acid Sequence↗
PubMed1987

Transport of the murine Mx protein into the nucleus is dependent on a basic carboxy-terminal sequence.

Cytoplasmic microinjection of murine Mx mRNA synthesized in vitro or nuclear microinjection of Mx cDNA under the control of a constitutive promoter into murine Mx- cells led to the accumulation of Mx protein in the nucleus and inhibited the replication of influenza virus but not of vesicular stomatitis virus (VSV). Similar results were also found with dog, rat, chicken, and monkey cells. A human lung fibroblast cell line (A549) was exceptional in that Mx protein was located predominantly in the cytoplasm and showed antiviral activity. Truncation of the 19 last residues of murine Mx protein almost completely abolished accumulation of Mx protein in the nucleus; however the activity against influenza virus was at least partially retained. The truncated region contains a segment rich in basic amino acids, similar to that reported for several nuclear location signals.

Amino Acid Sequence↗
PubMed1986

Sequence requirements for splicing of higher eukaryotic nuclear pre-mRNA.

We determined the effect on splicing of 24 point mutations in the 5' and 3' splice region of the large rabbit beta-globin intron. In vitro, 3' AG mutations drastically reduce 5' cleavage and abolish splicing. In vivo, the same mutations elicit efficient splicing at a cryptic, rather than the correct, 3' splice site. In vitro, mutations at all but 2 positions of the consensus 5' splice region impair correct splicing and promote joining of exon 1 to exon 3. In vivo, the same mutations show no effect, except for those converting 5' GT to AT or GA, which cause accumulation of lariat intermediate in vitro and in vivo. We conclude that the 5' GT need not be conserved for 5' cleavage and that it plays an important role in cleavage and exon joining at the 3' splice site.

Animals↗
PubMed1986

Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene.

PrP 27-30 is the major protein in purified preparations of scrapie agent. An almost complete PrP cDNA was used to select PrP-related genomic clones from normal hamster DNA. The gene contains a noncoding exon of 56 to 82 bp and a 2 kb coding exon, separated by a 10 kb intron. Transcription initiates at the same multiple sites in vivo and in vitro. The promoter lacks a TATA box and contains three repeats of the sequence GCCCCGCCC, which resembles the Sp1 binding site found in "housekeeping" genes. The PrP coding sequence encodes a presumptive amino-terminal signal peptide. The primary structure of PrP encoded by the gene of a healthy animal does not differ from that encoded by a cDNA from a scrapie-infected animal, suggesting that the different properties of PrP from normal and scrapie-infected brains are due to post-translational events.

Animals↗
PubMed1986

Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus.

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.

Amino Acid Sequence↗
PubMed1986

A new approach to high sensitivity differential hybridization.

We describe a new approach to differential hybridization, designed to identify cDNA clones representing rare mRNA species. Duplicate filters carrying a library of cDNA from phorbolmyristate acetate (PMA)-induced EL-4 cells in lambda gt11 were hybridized with high concentrations of unlabeled, cloned, single-stranded cDNA from induced and control EL-4 cells, respectively. Plaques binding single-stranded cDNA were revealed by a second round of hybridization with 35S-labeled DNA complementary to the vector moiety of the single-stranded cDNA. Plaques corresponding to PMA-induced mRNAs occurring at a level of about 1 part in 15,000 were isolated. We believe the method is at least ten times more sensitive than conventional differential hybridization.

Animals↗