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Biomedical subjects

B Dobberstein

Publications and source records attributed to B Dobberstein.

At least 73 records · Page 4Linked to original sources
PubMed1986

Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length.

We investigated the arrest of mRNA translation at predetermined sites by oligodeoxynucleotides complementary to defined coding sequences within a mRNA. An in vitro transcription and a wheat germ cell-free translation system were used for the synthesis of mRNA and protein, respectively. Oligodeoxynucleotides (10-, 15- and 20-mer) arrested polypeptide synthesis in a concentration-dependent manner at the site of their hybridization to the mRNA, as judged by the size of the translation products. A 5-mer oligodeoxynucleotide did not prevent synthesis of the full length protein. Ribosomes arrested by an oligodeoxynucleotide transiently stacked up and eventually disassembled. Upon dissociation of the ribosomes from the blocked site, nascent chains were released as peptidyl-tRNAs which in turn became rapidly converted to free polypeptide chains. None of these results was affected by the position within the reading frame to which the 3' end of the oligodeoxynucleotide hybridized. The general applicability of translation arrest by oligodeoxynucleotides was demonstrated for different mRNAs. Only partial arrest of translation was obtained when oligodeoxynucleotides were used to arrest translation in the reticulocyte cell-free system.

Animals↗
PubMed1986

Signal recognition particle-dependent membrane insertion of mouse invariant chain: a membrane-spanning protein with a cytoplasmically exposed amino terminus.

Invariant (Ii) chain is a membrane-spanning protein that is found associated intracellularly with class II histocompatibility antigens. In the endoplasmic reticulum Ii chain spans the membrane and exposes the NH2 terminus on the cytoplasmic and the COOH terminus on the lumenal side. This orientation across the membrane is demonstrated directly with the monoclonal antibody In-1, which exclusively recognizes the NH2 terminal cytoplasmically exposed part of Ii chain. Membrane insertion of Ii chain requires signal recognition particle and docking protein. When tested in a wheat germ cell free system, signal recognition particle arrests translation of Ii chain. No signal sequence is cleaved from Ii chain upon membrane insertion.

Amino Acid Sequence↗
PubMed1985

Translation efficiency of zein mRNA is reduced by hybrid formation between the 5'- and 3'-untranslated region.

The secondary structure of zein mRNA affects its translation potential. Here we show that in a cell-free system the translation efficiency of zein mRNA containing inverted repeats in the 5'- and 3'-untranslated regions is reduced. This translational block is released after deletion of the 3'-inverted repeat. We conclude that the translational block is caused by hybrid formation between the two inverted repeats. The translational efficiency of zein mRNAs, is also affected by varying the length or the primary structure of the 5'-untranslated region.

3' Untranslated Regions↗
PubMed1985

The gene encoding the Ia-associated invariant chain is located on chromosome 18 in the mouse.

The chromosomal assignment of the gene encoding the invariant (Ii) chain associated with the mouse immune response antigens (Ia) was determined by Southern blot analysis of DNA from a panel of mouse X Chinese hamster somatic cell hybrids cleaved with Hind III or Eco RI. Using a mouse li cDNA as a hybridization probe, we localized the gene coding for the invariant chain to mouse chromosome 18.

Animals↗
PubMed1984

A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences.

A system is described which permits the efficient synthesis of single proteins in vitro. The essential element in this expression system is a strong promoter derived from coliphage T5 which produces, with high efficiency, specific RNAs in capped or uncapped form, depending upon the experimental conditions used. The transcription-coupled capping of RNA allows the direct translation of the RNA in eukaryotic extracts from wheat germ as well as from HeLa cells. The synthesis of three different proteins is reported, including lysozyme, which is shown to be translocated across membranes when appropriate assay conditions are used. The simplicity of the experimental procedure, the high purity and specific activity of the [35S]methionine-labelled proteins produced offer a number of possibilities for the study of structure-function relationships of proteins.

Cell-Free System↗
PubMed1984

Structure of the murine Ia-associated invariant (Ii) chain as deduced from a cDNA clone.

The invariant (Ii) chain is a membrane-spanning glycoprotein found intracellularly associated with class II major histocompatibility complex (MHC) molecules. Using hybrid-selected translation and the Ii-specific monoclonal antibody In-1, we have isolated a cDNA clone (pIi-5) coding for most of the Ii chain. Sequence analysis of this clone reveals an open reading frame encoding 169 amino acid residues. The protein is rich in methionine and contains two potential N-glycosylation sites. No stretch of uncharged amino acid residues, characteristic for a membrane-spanning segment, is found close to the COOH-terminal end. There is one, however, close to the NH2-terminal end. As it is know that approximately 20 amino acid residues of Ii chain are exposed on the cytoplasmic side, we conclude that the Ii chain spans the membrane exposing the NH2 terminus on the cytoplasmic side and the COOH terminus on the luminal side.

Amino Acid Sequence↗
PubMed1983

The organization of the 7SL RNA in the signal recognition particle.

Digestion of the signal recognition particle (SRP) of dog pancreas with micrococcal nuclease results in the stepwise cleavage of the 300 nucleotide 7SL RNA moiety producing five major fragments approximately 220 (1), 150 (2), 72 (3), 62 (4) and 45 (5) nucleotides long. The RNA molecule is initially cut once yielding fragments 1 and 3. Further degradation releases fragments 2, 4 and 5. The introduction of the first nick into the 7SL RNA does not alter the structure nor the function of the SRP. Further degradation of the RNA results in disruption and loss of activity of the particle. The sequence of the RNA fragments shows that the nuclease causes discrete cuts in the RNA with minimal nibbling indicating that only few sites are accessible to the action of the enzyme. The five major products of nuclease digestion together span almost the entire length of the 7SL RNA. Nicking occurs mainly around the boundary region between the central S sequence and the flanking Alu sequences constituting the 7SL RNA (1). The S fragment is bound to the four largest polypeptides while the 5' and 3' Alu fragments are associated with the two smallest protein constituents of the SRP.

Animals↗
PubMed1983

Mouse histocompatibility genes: structure and organisation of a Kd gene.

The gene coding for the mouse H-2Kd antigen has been isolated by using a K-locus specific cDNA probe. The complete nucleotide sequence of the gene reveals eight exons separated by seven introns. Transcriptionally important DNA sequences (CCAAT and TATA) precede the first exon. Comparison with other H-2 genes shows extensive homology in exons as well as in introns. Two cDNA clones encoding Kd antigens have been analysed and provide evidence for at least two expressed Kd genes in the DBA/2 mouse. Comparison of the Kd antigen sequence to three other H-2 antigens indicates that gene conversion mechanism(s) act on H-2 genes. Analyses of exon donor and acceptor sites of different H-2 genes and cDNAs show that alternative splicing sites are used by different genes.

Alternative Splicing↗
PubMed1982

Structure and biosynthesis of histocompatibility antigens (H-2, HLA).

Histocompatibility antigens (H-2K, D and L, and HLA-A, B and C) are highly polymorphic cell surface proteins. Their primary structure has been determined by sequencing the protein, complementary DNAs (cDNAs) or genes in several laboratories. H-2Ld and Kd antigens are encoded by eight separate exons: one encodes the signal sequence, three encode the external domains, one encodes the membrane spanning segment and three encode the cytoplasmic domain. A similar structural organization has been found for an HLA gene. H-2 and HLA antigens are synthesized on membrane-bound ribosomes and are co-translationally inserted into the membrane of the endoplasmic reticulum. Here they assemble with beta 2-microglobulin, a small secretory protein. We describe the structure, the membrane insertion in vitro and in vivo, the intracellular transport and the surface expression of these antigens.

Animals↗
PubMed1982

Identification of an H-2Kd gene using a specific cDNA probe.

A cDNA clone known to code for a mouse histocompatibility (class I) antigen was found to contain a sequence specific for a subpopulation of H-2 genes. This unique sequence is located in the 3' non-coding region close to the stretch of poly(A) nucleotides. A subclone containing this fragment (pH-2d-5) has been used to select hybridizing mRNA. Translation of the mRNA in vitro shows that H-2Kd mRNA is selected. Southern blot analysis of DNA from congenic recombinant mice show that at least one gene containing this sequence is located at the K locus (region) of the major histocompatibility complex. This gene contains a 3.7-kb BglII and a 13-kb EcoRI restriction endonuclease fragment. This gene has been isolated from a genomic DNA library.

Animals↗
PubMed1982

Membrane insertion and oligomeric assembly of HLA-DR histocompatibility antigens.

HLA-DR histocompatibility antigens are assembled in the endoplasmic reticulum. This assembly has been studied in vitro and in vivo. Three polypeptides are involved in forming the oligomeric structure of HLA-DR antigens, DR alpha chains (molecular weight 35,000), DR beta chains (molecular weight 29,000) and DR gamma chains (molecular weight 33,000). They are cotranslationally inserted into the membrane of the endoplasmic reticulum, and all span the membrane. The size of the cytoplasmic portion of DR alpha and DR beta is about 500- 1000 daltons, whereas that of the DR gamma chain is about 3000 daltons. Oligomeric assembly of DR alpha, DR beta and DR gamma chains occurs shortly after their synthesis in the endoplasmic reticulum. DR gamma chains are synthesized in excess of DR alpha and DR beta chains, and hence in the endoplasmic reticulum they are found either in a complex with DR alpha and DR beta or in a free form. Free DR gamma chains remain in the endoplasmic reticulum, whereas DR gamma chains present in the oligomeric complex with DR alpha and DR beta undergo intracellular transport. Their molecular weight increases during transport, probably because of the addition of complex sugars in the Golgi complex. This is followed by the detachment of DR gamma chains from the oligomeric complex and the appearance of DR alpha and DR beta chains on the cell surface. Whether any DR gamma chains appear on the cell surface is uncertain.

Biological Transport↗
PubMed1982

Isolation and identification of a cDNA clone corresponding to an HLA-DR antigen beta chain.

The HLA-D locus in the major histocompatibility complex controls the expression of the genetically polymorphic HLA-DR antigens. mRNA coding for the beta chains of these antigens was partially purified from the human lymphoblastoid cell line Raji. The mRNA was copied into double-stranded cDNA and cloned in Escherichia coli. One clone, pDR-beta-1, obtained by hybrid selection, carries a 1070-base-pair insert comprising all of the coding region except the signal sequence and a substantial portion of the untranslated region. To identify pDR-beta-1, highly purified HLA-DR antigen beta chains derived from Raji cells were subjected to NH2-terminal amino acid sequence determination. This sequence displayed extensive homology with that deduced from the nucleotide sequence at the 5' end of the pDR-beta-1 coding region. Taken together, the amino acid and nucleotide sequences strongly argue in favor of Raji cells containing at least two beta-chain loci.

Amino Acid Sequence↗
PubMed1982

Characterization of molecules involved in protein translocation using a specific antibody.

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane-bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.

Animals↗