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W Bannwarth

Publications and source records attributed to W Bannwarth.

31 records · Page 2Linked to original sources
PubMed1991

Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an Escherichia coli expression system.

A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release. The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM.

Amino Acid Sequence↗
PubMed1990

Identical genes for trimethoprim-resistant dihydrofolate reductase from Staphylococcus aureus in Australia and central Europe.

The nucleotide sequence of a 1.25 kb BglII/EcoRI fragment from the 34 kb trimethoprim (Tp)-resistant plasmid pABU1 of Staphylococcus aureus 157/4696, isolated in Zürich, was determined. It contained the entire Tp-resistant dihydrofolate reductase gene, 197 bp of the thymidylate synthetase, 395 bp of a truncated gene and 111 bp of IS257R1. With the exception of one single base pair at position of 862 the sequence of the whole fragment was identical to nucleotides 1633 to 2885 of the Tp-resistant transposon Tn4003 in plasmid pSK1 from an Australian S. aureus isolate. This suggests the worldwide dissemination of Tn4003 in multiresistant Staphylococci.

Amino Acid Sequence↗
PubMed1990

New B cell epitopes in the Plasmodium falciparum malaria circumsporozoite protein.

Most antibodies directed against the Plasmodium falciparum circumsporozoite (CS) protein react with its central domain, which contains about 40 repeats of the tetrapeptide Asn-Ala-Asn-Pro (NANP). To search for new epitopes in the non-repetitive part of the CS protein, we expressed the non-repetitive regions of the protein in E. coli as fusion proteins with mouse dihydrofolate reductase linked to six adjacent histidine residues. These fusion proteins were obtained at greater than 70% purity by a single Ni-chelate affinity chromatography step. Of the new epitopes defined in the C-terminal portion of the CS protein, three are located in a stretch of 65 amino acids immediately C-terminal of the protein's central repetitive domain. Pooled sera from inhabitants of a malaria-endemic area reacted with epitopes in this region of the molecule, and four mouse monoclonal antibodies to this region also reacted with the native CS protein on sporozoites. Two of the monoclonal antibodies reacted with a peptide PNDPNRNVD derived from a conserved region of the CS protein. The other two antibodies showed different reactivities to sporozoites of the NF54 and Ro59 parasite isolates. One, which reacted with a peptide ENANANNAV, recognized Ro59 but not NF54 sporozoites, while the other reacted with a small percentage of NF54 but not Ro59 sporozoites. Antibodies which react with non-repetitive regions of the CS protein could contribute to maintaining its genetic variability.

Amino Acid Sequence↗
PubMed1988

A single 12.5-kilobase androgen-regulated mRNA encoding multiple proline-rich polypeptides in the ventral prostate of the rat.

Synthetic 32P-labeled oligonucleotides have been used to identify the prostatic proline-rich polypeptide (PRP) mRNA which has partially been characterized. The 14-mer d(G-G-T-T-C-T-G-C-A-T-A-A-T-G) complementary to the coding sequence for His-Tyr-Ala-Glu-Pro, a sequence element occurring in all 38-residue PRP variants, hybridizes specifically with a 12.5-kilobase mRNA which is clearly androgen-controlled. This oligonucleotide was used as an efficient primer for the construction of a PRP-specific lambda gt10 cDNA library. The nucleotide sequence of the inserts from several recombinant clones has been determined. This structural analysis revealed a PRP mRNA encoding a large precursor containing a number of tandemly repeated units. Each repeat codes for a sequence of 100 amino acids in which the highly conserved PRP sequence is embedded. From this polyprotein the large number of PRP variants must be generated by a post-translational processing mechanism which is still unknown. The high degree of conservation of both nucleotide and amino acid sequence in the entire unit also indicates that the PRP gene(s) likely evolved by multiplication of a 300-base pair ancestral DNA sequence. This has resulted in a noninterrupted repetitive DNA coding segment which is detected at the genomic level.

Amino Acid Sequence↗
PubMed1988

Association of degradation and secretion of three chimeric polypeptides in Escherichia coli.

We investigated the stability of fusion proteins composed of the signal peptide of the heat-labile enterotoxin of Escherichia coli and three polypeptides: the bacterial cytoplasmic chloramphenicol acetyltransferase, the mouse dihydrofolate reductase, and human immune interferon. We demonstrate that these proteins are rapidly degraded as a result of being targeted to the secretion apparatus of E. coli, with the extent of degradation varying among the three fusion proteins. Four lines of experimental evidence are presented in support of this suggestion. First, the chimeric polypeptides containing a functional signal peptide were detected in low amounts in vivo. When a mutation was introduced in the signal peptide, resulting in lack of recognition by the secretion apparatus, the chimeric proteins accumulated at high levels in the cytoplasm of the cell. Second, both the wild-type and mutant polypeptides accumulated in a purified and reconstituted in vitro translation system from E. coli and were equally susceptible to digestion by an exogenous protease. Third, the chimeric polypeptides lacking the signal peptide accumulated in a stable form in vivo. Fourth, the precursors of the proteins containing a functional signal peptide accumulated in a secA ts mutant at the restrictive temperature when secretion was blocked, suggesting that degradation is tightly linked to the secretion apparatus.

Acetyltransferases↗
PubMed1987

Dideoxy sequencing of double-stranded DNA from poly(A)-extended 3'-ends.

A simple method has been developed for sequencing double-stranded DNA by the chain termination method. The DNA to be sequenced is cut with a restriction enzyme that leaves a 3'-overhang which is extended by terminal deoxynucleotidyltransferase with limiting amounts of dATP. The sequencing reaction is then primed with an oligo(dT) primer which has a base pair "anchor" complementary to the overhang generated by the restriction enzyme. The method presented here eliminates the need for subcloning of the DNA or sequencing by chemical modification. Furthermore, sequences of more than 300 nucleotides are obtained from any 3'-overhanging restriction end.

Autoradiography↗
PubMed1986

Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation.

We have characterized the vaccinia virus 11-kd late promoter through 5' and 3' deletions and site-directed mutagenesis. The promoter function appears to be contained within an approximately 30-bp fragment, which after translocation is able to direct RNA synthesis late in infection at a reduced level. We demonstrate that a TAAAT sequence in the proximal part of the promoter is essential for its function. This cis-acting element is highly conserved within vaccinia virus late promoters and overlaps the site of transcription initiation. Deletions or mutations within this conserved element completely inactivate the promoter. The evidence indicates that the TAAAT motif functions as a TATA box. The region immediately upstream of the TAAAT motif determines the promoter strength.

Base Sequence↗
PubMed1986

Subregions of a conserved part of the HIV gp41 transmembrane protein are differentially recognized by antibodies of infected individuals.

A 240-bp DNA fragment encoding a peptide, designated ENV(80), homologous to a conserved part of the gp41 transmembrane glycoprotein of human immunodeficiency virus (HIV) was chemically synthesized and inserted into different plasmid expression vectors. Escherichia coli transformants containing these plasmid constructs produced upon induction high amounts of either an ENV(80) peptide of relative molecular mass (Mr) of 10,000 or the same ENV(80) peptide N-terminally fused to E. coli chloramphenicol acetyltransferase (CAT) or to mouse dihydrofolate reductase (DHFR) having Mr of 36,000 and 31,000 respectively. All polypeptides containing the ENV(80) sequences were strongly reactive with antibodies present in sera from AIDS virus-infected individuals, but not with control sera. The strategy of gene assembly allowed the expression of ENV(80) subfragments fused to DHFR. The serodiagnosis of 15 positive sera by Western blot analysis using these bacterially synthesized ENV(80) subfragments revealed the presence of several immunoreactive epitopes on the 80-amino acid polypeptide which were recognized differently by the various patients.

Acquired Immunodeficiency Syndrome↗
PubMed1986

A system for the simultaneous chemical synthesis of different DNA fragments on solid support.

A mechanical apparatus for the simultaneous synthesis of different DNA fragments on solid support is described. This new system allows the use of different kinds of supports and is amenable to automation. Utilizing this apparatus the synthesis of DNA fragments can be speeded up considerably. The effectiveness of the system is demonstrated by the simultaneous synthesis of DNA fragments up to 36 nucleotides long using phosphoamidites as building blocks and controlled pore glass as solid support.

Automation↗
PubMedDate not supplied

The gene encoding the T-cell receptor alpha-chain maps close to the Np-2 locus on mouse chromosome 14.

Serological and molecular genetic analyses of T-cell clones have shown that the T-cell antigen receptor apparently comprises two glycosylated, disulphide-linked polypeptide chains (alpha and beta), both of which span the cell membrane. Cloning of the genes encoding the two chains from mouse and human DNA has shown that the alpha- and beta-chains are composed of variable (V) and conserved (C) regions in agreement with peptide mapping data. Gene segments encoding variable and conserved domains of the beta-chain have been identified and undergo rearrangements during T-cell differentiation. The genes encoding the alpha-chain, so far described at the level of complementary DNA clones, also identify DNA rearrangements. Thus, the genes encoding the T-cell receptor show the same structure and dynamic behaviour as immunoglobulin genes, indicating that the two gene families belong to the same supergene family; this evolutionary relationship is supported by the fact that the genes encoding the beta-chain of the T-cell receptor are closely linked to immunoglobulin kappa light-chain genes on chromosome 6 in mouse. In man, however, the beta genes map to chromosome 7 (ref. 14) whereas the kappa-chain genes are located on chromosome 2, indicating that linkage between the two gene families is not needed for proper expression. Here we describe genomic clones encoding the constant portion of the T-cell receptor alpha-chain and map the gene to chromosome 14 in mouse, close to the gene for purine nucleoside phosphorylase (Np-2) which, in man, has been associated with T-cell immunodeficiencies.

Animals↗
PubMedDate not supplied

Compensatory mutations suggest that base-pairing with a small nuclear RNA is required to form the 3' end of H3 messenger RNA.

Processing of the 3' end of sea urchin H3 histone pre-mRNA requires conserved sequence elements and the presence of U7 snRNA. A mutation in the conserved CAAGAAGA sequence of the H3 pre-mRNA that renders 3' processing of this precursor defective is shown to be suppressed by a compensatory change in the U7 snRNA, restoring the base-pairing potential of the two RNAs. RNA-RNA contacts between these two molecules appear to be an essential feature of the 3' processing reaction.

Animals↗