Search PubMed⌕ Search

Biomedical subjects

C Weissmann

Publications and source records attributed to C Weissmann.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1985

A 46-nucleotide promoter segment from an IFN-alpha gene renders an unrelated promoter inducible by virus.

A hybrid gene consisting of the human IFN-alpha 1 promoter and a beta-globin transcription unit is expressed correctly only after viral induction. To determine the region required for inducibility, 25 hybrid promoters consisting of varying upstream IFN-alpha 1 and downstream beta-globin promoter moieties were analyzed, and 5'-deletion analysis was performed on an inducible hybrid promoter. An IFN promoter region from position -109 to -64 conferred maximal inducibility on downstream beta-globin promoter segments and even on the intact beta-globin promoter. This region is strikingly conserved among human IFN-alpha and -beta genes. As constitutive expression of the beta-globin gene was not diminished by placing IFN promoter fragments in various positions, induction is attributed largely to positive, rather than to negative control.

Animals↗
PubMed1985

Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes.

Mutants of the rabbit beta-globin gene lacking the natural site of branch formation in the second intervening sequence have been analyzed for in vitro splicing activity. RNAs transcribed from these mutants were spliced, via lariat formation, at a reduced rate compared to wild-type RNA. The sites of branch formation were mapped by direct RNA analysis and primer-extension analysis. The sequences at the branch sites in the three mutants examined did not conform to the previously determined consensus sequence, nor were the 5' splice sites and branch sites complementary.

Animals↗
PubMed1984

The expression of human interferon alpha genes.

We have determined the levels of mRNAs for IFN-beta, IFN-gamma and various alpha-IFNs (IFN-alpha 1, -alpha 2, -alpha 4, -alpha 5, -alpha 6, -alpha 7, -alpha 8 and -alpha 14) in normal and leukaemic human blood leucocytes and several cell lines induced in different fashions. The ratio of alpha to beta IFN transcripts varied greatly, depending on the cell type. The levels of the individual IFN-alpha RNAs were very different: IFN-alpha 1, -alpha 2 and -alpha 4 RNAs constituted the major fraction of the IFN-alpha transcripts measured, while IFN-alpha 6, -alpha 7, -alpha 8 and -alpha 14 were minor components in normal, induced leucocytes. Moreover, there was a striking difference in the proportion of individual IFN-alpha mRNA species in different cell types, in particular between normal and leukaemic cells; for example all cases of myeloblastic leukaemia examined showed a high expression of IFN-alpha 14. Use of different induction protocols did not significantly affect the proportion of IFN mRNAs. Analysis of the human IFN-alpha 1 gene by reversed genetics led to the identification of a segment of 5' flanking sequence between positions 117 and 68 upstream of the cap site which is required for inducibility by virus.

Cell Line↗
PubMed1984

Differential expression of human interferon genes.

We developed a method for quantitating closely related mRNAs by S1 mapping and used it to determine the levels of mRNAs for IFN-beta, IFN-gamma and various alpha IFNs (IFN-alpha 1, -alpha 2, -alpha 4, -alpha 5, -alpha 6, -alpha 7, -alpha 8 and -alpha 14) in human peripheral blood leukocytes, lymphoblastoid (Namalwa), HeLa and human fibroblastic cells, induced in different fashions. The ratio of alpha to beta IFN transcripts varied greatly, depending on the cell type. The levels of the individual IFN-alpha RNAs were very different: IFN-alpha 1, -alpha 2 and -alpha 4 RNAs constituted the major fraction of the IFN-alpha transcripts measured. Moreover, there was a striking difference in the proportion of individual IFN-alpha mRNA species in different cell types. Use of different induction protocols did not significantly affect the proportion of IFN mRNAs. IFN production was not proportional to mRNA level in all cases, as lymphoblastoid cells induced by incubation at high density and virus-induced HeLa cells contained high levels of IFN-beta but produced little antiviral activity.

Burkitt Lymphoma↗
PubMed1984

The chromosomal location of mouse interferon alpha genes.

The chromosomal location of mouse leukocyte-interferon (IFN-alpha) genes was determined by Southern blot analysis of DNA from a panel of Chinese hamster x mouse somatic cell hybrids using a mouse IFN-alpha cDNA as a hybridization probe. All resolvable mouse genes are located on mouse chromosome 4. In addition, two common restriction site polymorphisms within these genes were identified in several mouse strains.

Animals↗
PubMed1984

Two non-allelic human interferon alpha genes with identical coding regions.

A hitherto undescribed human interferon-alpha (IFN-alpha) gene, IFN-alpha 13, has been isolated and characterized. Its entire coding sequence is completely identical with that of IFN-alpha 1; the 5' and 3' non-coding regions differ by 4.5% and 3.8%, respectively. As the two genes are not allelic, we conclude that the similarity is due to a very recent gene conversion event. Both genes are expressed in virus-induced leukocytes.

Base Sequence↗
PubMed1984

Modular structure of the beta-globin and the TK promoters.

Three regions important for transcription, the ATA box, the middle and the distal element, have been identified in the 5'-flanking region of both the rabbit beta-globin and herpes simplex virus thymidine kinase (TK) gene. To determine whether these elements are functionally equivalent, we constructed mosaic promoters containing all combinations of the three regions from both promoters and joined them to the beta-globin transcription unit. In an enhancer-dependent transient expression assay the beta-globin, the TK and all mosaic promoters retaining the beta-globin ATA box were about equally active, however, mosaic promoters with the TK ATA box were 4- to 10-fold less active. We conclude that homologous elements are, in principle, exchangeable and suggest an explanation why certain combinations of elements function poorly.

Animals↗