Markus Aebi

cDNA structures and regulation of two interferon-induced human Mx proteins.

Human cells treated with interferon synthesize two proteins that exhibit high homology to murine Mx1 protein, which has previously been identified as the mediator of interferon-induced cellular resistance of mouse cells against influenza viruses. Using murine Mx1 cDNA as a hybridization probe, we have isolated cDNA clones originating from two distinct human Mx genes, designated MxA and MxB. In human fibroblasts, expression of MxA and MxB is strongly induced by alpha interferon (IFN-alpha), IFN-beta, Newcastle disease virus, and, to a much lesser extent, IFN-gamma, MxA and MxB proteins have molecular masses of 76 and 73 kilodaltons, respectively, and their sequences are 63% identical. A comparison of human and mouse Mx proteins revealed that human MxA and mouse Mx2 are the most closely related proteins, showing 77% sequence identity. Near their amino termini, human and mouse Mx proteins contain a block of 53 identical amino acids and additional regions of very high sequence similarity. These conserved sequences are also present in a double-stranded RNA-inducible fish gene, which suggests that they may constitute a functionally important domain of Mx proteins. In contrast to mouse Mx1 protein, which accumulates in the nuclei of IFN-treated mouse cells, the two human Mx proteins both accumulate in the cytoplasm of IFN-treated cells.

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.

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.

STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo

N‐linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid‐linked core‐oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid‐linked core‐oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N‐linked glycosylation. Besides novel mutants deficient in the assembly of the lipid‐linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3–3 cells very little glycosyl transfer occurs from incomplete lipid‐linked oligosaccharide, whereas the transfer of full‐length Glc3Man9GlcNAc2 is hardly affected as compared with wild‐type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex.

The yeast WBP1 is essential for oligosaccharyl transferase activity in vivo and in vitro.

Asparagine‐linked N‐glycosylation is a highly conserved and functionally important modification of proteins in eukaryotic cells. The central step in this process is a cotranslational transfer of lipid‐linked core oligosaccharides to selected Asn‐X‐Ser/Thr‐sequences of nascent polypeptide chains, catalysed by the enzyme N‐oligosaccharyl transferase. In this report we show that the essential yeast protein WBP1 (te Heesen et al., 1991) is required for N‐oligosaccharyl transferase in vivo and in vitro. Depletion of WBP1 correlates with a defect in transferring core oligosaccharides to carboxypeptidase Y and proteinase A in vivo. In addition, in vitro N‐glycosylation of the acceptor peptide Tyr‐Asn‐Leu‐Thr‐Ser‐Val using microsomal membranes from WBP1 depleted cells is reduced as compared with membranes from wild‐type cells. We propose that WBP1 is an essential component of the oligosaccharyl transferase in yeast.

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.

Yeast Wbp1p and Swp1p form a protein complex essential for oligosaccharyl transferase activity.

Asparagine‐linked N‐glycosylation is an essential protein modification occurring in all eukaryotic cells. The central step is the co‐translational transfer of the core oligosaccharide assembled on the lipid carrier dolichol phosphate to selected Asn‐X‐Ser/Thr residues of nascent polypeptide chains in the endoplasmic reticulum. This reaction is catalyzed by the enzyme N‐oligosaccharyl transferase. In yeast, Wbp1p is an essential component of this enzyme. Using a high copy number suppression approach, the SWP1 gene was isolated as an allele specific suppressor of a wbp1 mutation. Swp1p is a 30 kDa type I transmembrane protein and essential for cell viability. Similar to Wbp1p, depletion of Swp1p results in reduced N‐oligosaccharyl transferase activity in vivo and in vitro. Wbp1p and Swp1p can be chemically cross‐linked, suggesting that both proteins are essential constituents of the N‐oligosaccharyl transferase complex.

The STT3 protein is a component of the yeast oligosaccharyltransferase complex

Abstract N-linked protein glycosylation is an essential process in eukaryotic cells. In the central reaction, the oligosaccharyltransferase (OTase) catalyzes the transfer of the oligosaccharide Glc3Man9GlcNAc2 from dolicholpyrophosphate onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum. The product of the essential gene STT3 is required for OTase activity in vivo, but is not present in highly purified OTase preparations. Using affinity purification of a tagged Stt3 protein, we now demonstrate that other components of the OTase complex, namely Ost1p, Wbp1p and Swp1p, specifically co-purify with the Stt3 protein. In addition, different conditional stt3 alleles can be suppressed by overexpression of either OST3 and OST4, which encode small components of the OTase complex. These genetic and biochemical data show that the highly conserved Stt3p is a component of the oligosaccharyltransferase complex.

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation.

SummaryMutations in the PRP20 gene of yeast show a pleiotropic phenotype, in which both mRNA metabolism and nuclear structure are affected. srm1 mutants, defective in the same gene, influence the signal transduction pathway for the pheromone response. The yeast PRP20/SRM1 protein is highly homologous to the RCC1 protein of man, hamster and frog. In mammalian cells, this protein is a negative regulator for initiation of chromosome condensation. We report the analysis of two, independently isolated, recessive temperature-sensitive prp20 mutants. They have identical G to A transitions, leading to the alteration of a highly conserved glycine residue to glutamic acid. By immunofluorescence microscopy the PRP20 protein was localized in the nucleus. Expression of the RCC1 protein can complement the temperature-sensitive phenotype of prp20 mutants, demonstrating the functional similarity of the yeast and mammalian proteins.

Model formulation support in risk management

Risk management is a broad field that integrates many disciplines to tackle issues of different nature. Consequently, many different modeling paradigms have been proposed for problem solving and decision making in risk management. The purpose of this paper is to show that despite their differences, the modeling paradigms can be accommodated into one generic model formulation process that consists of: (i) problem structuring, (ii) problem formalization, and (iii) problem resolution. We will show this for the most frequently used modeling paradigms in risk management: (i) decision modeling, (ii) data/knowledge modeling, and (iii) dynamic modeling. The benefits of this generic modeling approach are many. Most important is that the risk analyst can focus more on the problem content than on analytical issues which ultimately helps in the communication to the decision maker. The theoretical discussion will be accompanied by representative examples from the field of risk management. The paper closes with a discussion and a research agenda concerning model formulation support in risk management.