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J A Lenstra

Publications and source records attributed to J A Lenstra.

At least 73 records · Page 4Linked to original sources
PubMed1990

Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus.

The amino acid sequences recognized by monoclonal antibodies (MAbs) specific for the antigenic site IV of the spike protein S of transmissible gastroenteritis virus were analyzed by PEPSCAN. All MAbs of group IV recognized peptides from the S region consisting of residues 378 to 390. In addition, the neutralizing MAbs (subgroup IV-A) also bound to peptides from the region consisting of residues 1173 to 1184 and to several other peptides with a related amino acid composition. The contribution of the individual residues of both sequences to the binding of a MAb was determined by varying the length of the peptide and by a consecutive deletion or replacement of parental residues by the 19 other amino acids. The sequence consisting of residues 326 to 558, tested as part of a cro-beta-galactosidase hybrid protein, was antigenic, but the sequence consisting of residues 1150 to 1239 was not. Furthermore, antibodies raised in rabbits against the peptide SDSSFFSYGEIPFGN (residues 377 to 391), but not those raised against the peptide VRASRQLAKDKVNEC (residues 1171 to 1185), recognized the virus and had neutralizing activity. We infer that the epitope of the neutralizing MAbs is composite and consists of the linear sequence SFFSYGEI (residues 380 to 387) with contributions of A, D, K, N, Q, or V residues from other parts of the S molecule. The complex epitope was simulated by synthesizing peptides in which the sequences consisting of residues 380 to 387 and 1176 to 1184 were combined. MAbs of subgroup IV-A recognized the combination peptides two to six times better than the individual sequences. These results may offer prospects for the development of an experimental vaccine.

Amino Acid Sequence↗
PubMed1989

Analysis of an immunodominant region of infectious bronchitis virus.

We analyzed the antigenic fine-structure of an immunodominant region in the peplomer protein of infectious bronchitis virus. This region near the N-terminus of the S2 subunit is recognized by polyclonal antisera and by the majority of mAb that cross-react with denatured protein. Despite their involvement in neutralization, epitopes in this region were conserved in different serotypes. Epitopes of four mAb and two chicken antisera were localized by using prokaryotic expression of cDNA fragments, and overlapping peptides with lengths increasing from 3 to 12 residues (PEPSCAN). We found overlapping epitopes with lengths of 6, 9, 11, and more than 17 residues. The results indicate that the expression products are antigenically equivalent to denatured protein fragments. This suggests a general strategy for the localization of sequential epitopes in large proteins. We propose that the immunodominance of the N-terminal region of S2 is explained by features of the protein structure. Presumably, this region is a protruding protein segment of about 20 residues with a high local mobility, as indicated by the antigenicity of the peptides. The conservation of the sequence points to an involvement in a molecular recognition process during infection.

Amino Acid Sequence↗
PubMed1989

Phylogeny of antigenic variants of avian coronavirus IBV.

The sequences of the peplomeric S1 protein of four serologically distinct strains of the infectious bronchitis virus (IBV), an avian coronavirus, have been determined. The S1 protein is thought to contain the serotype-specific neutralization epitopes and to be the main target of antigenic variation. An alignment with sequences of three strains published previously showed that from the 545 amino acid residues only 243 have been conserved. Clustering of substitutions suggests that most serotype determinants are located within the first 300 amino acid residues of S1. A phylogenetic tree of the S1 sequences showed very variable rates of divergence. Differences in topology with a tree based on RNAse-T1 fingerprint data indicate that some of the IBV strains have arisen by genetic recombination.

Amino Acid Sequence↗
PubMed1989

Antigenicity of the peplomer protein of infectious bronchitis virus.

To study the antigenic structure of the peplomer protein of the avian coronavirus infectious bronchitis virus, fragments from the peplomer gene were generated by restriction-enzyme cleavage or by limited DNase digestion and inserted in the Escherichia coli expression plasmid pEX (Stanley and Luzio, 1984). The antigenicity of the expression products was tested using a number of polyclonal antisera and monoclonal antibodies. The polyclonal antisera recognized different sets of epitopes in the 1162-residue sequence. The N-terminal region of one of the two subunits, S2, was recognized by all polyclonal sera and by two monoclonal antibodies. This clearly immunodominant region contains at least two adjacent or overlapping epitopes, one of which has been localized within 18 residues. The epitopes found as antigenic pEX expression products do not coincide with the regions in the S1 subunit that have been found to contain hypervariable sequences. We suggest that these regions constitute conformation dependent neutralization epitopes that cannot be detected in the pEX system. The relevance of our findings for vaccine development is discussed.

Amino Acid Sequence↗
PubMed1987

Evidence for a coiled-coil structure in the spike proteins of coronaviruses.

The amino acid sequences of the spike proteins from three distantly related coronaviruses have been deduced from cDNA sequences. In the C-terminal half, an homology of about 30% was found, while there was no detectable sequence conservation in the N-terminal regions. Hydrophobic "heptad" repeat patterns indicated the presence of two alpha-helices with predicted lengths of 100 and 50 A, respectively. It is suggested that, in the spike oligomer, these alpha-helices form a complex coiled-coil, resembling the supersecondary structures in two other elongated membrane proteins, the haemagglutinin of influenza virus and the variable surface glycoprotein of trypanosomes.

Amino Acid Sequence↗
PubMed1987

cDNA cloning and sequence analysis of the gene encoding the peplomer protein of feline infectious peritonitis virus.

The peplomer gene of feline infectious peritonitis virus (FIPV) strain 79-1146 was isolated from a genomic cDNA library by differential hybridization with RNA 2 and 3 as probes. From the nucleotide sequence a primary translation product of 1452 residues (Mr 160,472) was predicted, containing an N-terminal signal sequence, a C-terminal transmembrane segment and 35 potential N-linked glycosylation sites. By S1 nuclease analysis the 5' end of the presumptive RNA 2 body was located at about 30 nucleotides upstream from the initiating AUG codon. At approximately the same position a nine nucleotide sequence ACUAAACUU was found, which was also present 37 nucleotides downstream from the open reading frame. Comparison of the sequences of the FIPV, murine hepatitis virus and infectious bronchitis virus peplomer proteins showed about 27% overall homology, with most conservation in the C-terminal half.

Amino Acid Sequence↗
PubMed1986

Predicted membrane topology of the coronavirus protein E1.

The structure of the envelope protein E1 of two coronaviruses, mouse hepatitis virus strain A59 and infectious bronchitis virus, was analyzed by applying several theoretical methods to their amino acid sequence. The results of these analyses combined with earlier data on the orientation and protease sensitivity of E1 assembled in microsomal membranes lead to a topological model. According to this model, the protein is anchored in the lipid bilayer by three successive membrane-spanning helices present in its N-terminal half whereas the C-terminal part is thought to be associated with the membrane surface; these interactions with the membrane protect almost the complete polypeptide against protease digestion. In addition, it is predicted that the insertion of E1 into the membrane occurs by the recognition of the internal transmembrane region(s) as a signal sequence.

Amino Acid Sequence↗
PubMed1986

Genes coding for the elongation factor EF-1 alpha in Artemia.

The elongation factor EF-1 alpha is one of the most abundant proteins in eukaryotic cells, where it catalyzes the binding of aminoacyl-tRNA to ribosomes. The genes coding for this protein in the brine shrimp Artemia were analyzed by gene cloning, electron microscopy and chromosomal blot hybridization. There are only a few (about four) copies of one type of gene per haploid genome. These genes contain five exons divided over 10(4) base pairs. Local rearrangements give rise to a number of gene variants. Cross-hybridizations of Artemia cDNA probes with yeast and Drosophila DNA revealed two different yeast EF-1 alpha genes and one or two different Drosophila genes, respectively. Nucleotide sequencing revealed signals for synthesis and processing of EF-1 alpha transcripts as well as the exact location of exons. One interruption in the coding sequence corresponds closely to a splice junction in the gene coding for the homologous chloroplast protein EF-Tu from Euglena gracilis, presumably of prokaryotic origin. The first exon in the chloroplast gene codes for the region of EF-Tu that is homologous to regions of the elongation factor EF-G and of the initiation factor IF2, respectively.

Animals↗
PubMed1986

The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison with Beaudette strains.

The amino acid sequence of the gene for the peplomer protein of the vaccine strain M41 and the Beaudette laboratory strain M42-Salk of avian infectious bronchitis virus (IBV) have been derived from cDNA sequences. As found with other coronaviruses, the peplomer protein carries the epitopes eliciting neutralizing antibodies. The gene encodes a primary translation product of 1162 amino acids with a molecular weight of 128,079. The use of a recent algorithm to predict membrane-protein interactions led to the unambiguous localization of the signal peptide and a transmembrane anchor alpha-helix at the C-terminus. At 50 positions amino acid differences were found between M41 and two Beaudette strains (M42-Salk and M42-Houghton). They are partly clustered in two regions of the protein. These two regions are candidates for neutralization epitopes of the protein.

Amino Acid Sequence↗
PubMed1985

Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia.

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.

Animals↗
PubMed1984

Extraction of SV40 T-antigen from lysates of transformed cells.

Subcellular fractions from SV-40 transformed hamster lens cells, prepared by chemical extractions, were tested for the presence of T-antigen by immunoautoradiography. Most of the T-antigen was present in the nucleus and was resistant to extraction by 2 M NaCl, indicating an association with the nuclear matrix. Another part of the T-antigen was, under certain conditions, resistant to extraction of the cells with a nonionic detergent. This T-antigen could be solubilized by Ca2+ at low temperature, conditions that also cause a specific depolymerization of microtubules.

Animals↗
PubMed1983

Topography of the total protein population from cultured cells upon fractionation by chemical extractions.

Chemical extractions are proposed as a major tool for a fractionation of cellular proteins. As a model system, proteins from cultured hamster lens cells have been divided by independent extractions into seven subcellular fractions, corresponding to water-soluble proteins and the proteins from membranes, microfilaments (and other deoxycholate-soluble proteins), intermediate filaments, microtubules, polysomes and nuclei respectively. The latter two fractions have been subfractionated yielding ribosomal proteins, the elongation and initiation factors of the protein-synthesis machinery, chromatin proteins and non-chromatin proteins. The protein compositions of the fractions have been analyzed by one-dimensional and two-dimensional gel electrophoresis. This resulted in an almost complete topography of the proteins detected on two-dimensional gels of total-cell lysates. Comparison of two-dimensional patterns of proteins from the total-cell lysate and proteins from hamster erythrocytes or from liver, muscle or brain tissue showed that the different cell types have only few proteins in common. Two proteins are common to all of these cell types, namely actin and a 68-kDa protein. The latter protein was, like actin, vimentin and the tubulin subunits, also present in most cell fractions. Evidence is presented that this protein is identical to a 68-kDa heat-shock protein.

Animals↗
PubMed1983

Genes for elongation factor EF-1 alpha in the brine shrimp Artemia.

A plasmid carrying a cDNA sequence coding for elongation factor EF-1 alpha from Artemia was used to probe blots of mRNA and chromosomal DNA from Artemia. A messenger length for EF-1 alpha corresponding to 1850 nucleotides was found. Southern blots pointed to a limited number (1-4) of genes, coding for EF-1 alpha. From an Artemia gene library a recombinant phage was isolated, which contains genomic sequences of EF-1 alpha. S1-nuclease mapping indicated the presence of intervening sequences within this cloned gene.

Animals↗