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Biomedical subjects

E Tischer

Publications and source records attributed to E Tischer.

17 recordsLinked to original sources
PubMed1996

Beta-amyloid precursor protein. Location of transmembrane domain and specificity of gamma-secretase cleavage.

The formation of beta-amyloid by processing of its precursor protein is a characteristic of Alzheimer's disease. Two proteolytic cleavages produce the amino and carboxyl termini of beta-amyloid, with the latter cleavage site located within the transmembrane domain. Using DNA mutagenesis, we investigated the membrane position and sequence requirements for carboxyl-terminal processing of the beta-amyloid domain. Substitution of negatively charged residues across positions 40-46 of the beta-amyloid domain precluded both beta-amyloid formation and precursor maturation associated with secretory protein transport. In contrast, identical substitutions from positions 48-50 had no adverse effects. Since charged residues typically prevent protein membrane insertion, these data define the membrane boundary to position 46/47, a location allowing greater access to carboxyl-terminal processing of beta-amyloid, possibly without membrane destruction. Deletions within the carboxyl-terminal domain, including 4 residues spanning positions 39-42 of beta-amyloid, resulted in formation of the beta-amyloid peptide. Substituting residues 38-47 or 39-56 of the beta-amyloid domain in the precursor with a transmembrane sequence from another protein yielded a approximately 4-kDa beta-amyloid peptide, reflecting a loose residue specificity for carboxyl-terminal processing to beta-amyloid.

Amino Acid Sequence↗
PubMed1996

Mapping protein-protein interactions by affinity-directed mass spectrometry.

A precise and rapid method for identifying sites of interaction between proteins was demonstrated; the basis of the method is direct mass spectrometric readout from the complex to determine the specific components of the proteins that interact--a method termed affinity-directed mass spectrometry. The strategy was used to define the region of interaction of a protein growth factor with a monoclonal antibody. A combination of proteolytic digestion and affinity-directed mass spectrometry was used to rapidly determine the approximate location of a continuous binding epitope within the growth factor. The precise boundaries of the binding epitope were determined by affinity-directed mass spectrometric analysis of sets of synthetic peptide ladders that span the approximate binding region. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule-molecule contacts, including ligand-receptor and protein-oligonucleotide interactions.

Amino Acid Sequence↗
PubMed1991

The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing.

Vascular endothelial growth factor (VEGF) is an apparently endothelial cell-specific mitogen that is structurally related to platelet-derived growth factor. By Northern blot and protein analyses, we show that VEGF is produced by cultured vascular smooth muscle cells. Analysis of VEGF transcripts in these cells by polymerase chain reaction and cDNA cloning revealed three different forms of the VEGF coding region, as had been reported in HL60 cells. The three forms of the human VEGF protein chain predicted from these coding regions are 189, 165, and 121 amino acids in length. Comparison of cDNA nucleotide sequences with sequences derived from human VEGF genomic clones indicates that the VEGF gene is split among eight exons and that the various VEGF coding region forms arise from this gene by alternative splicing: the 165-amino-acid form of the protein is missing the residues encoded by exon 6, whereas the 121-amino-acid form is missing the residues encoded by exons 6 and 7. Analysis of the VEGF gene promoter region revealed a single major transcription start, which lies near a cluster of potential Sp1 factor binding sites. The promoter region also contains several potential binding sites for the transcription factors AP-1 and AP-2; consistent with the presence of these sites, Northern blot analysis demonstrated that the level of VEGF transcripts is elevated in cultured vascular smooth muscle cells after treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate.

Amino Acid Sequence↗
PubMed1989

Vascular endothelial growth factor: a new member of the platelet-derived growth factor gene family.

Using applications of the polymerase chain reaction (PCR) technique, cDNA clones have been isolated encoding bovine vascular endothelial growth factor (VEGF), a mitogen with specificity for vascular endothelial cells. Analysis of the clones indicates that VEGF can exist in two forms, probably due to alternative RNA splicing. The amino acid sequences predicted from the clones also show that VEGF shares homologies of about 21% and 24% respectively with the A and B chains of human platelet-derived growth factor (PDGF), and has complete conservation of the eight cysteine residues found in both mature PDGF chains. The homology is not reflected in function, however, since the cell types responsive to VEGF are distinct from those responsive to homo- and heterodimers of the PDGF chains.

Amino Acid Sequence↗
PubMed1989

Structural analysis of the gene for human acidic fibroblast growth factor.

Genomic clones derived from the gene for human acidic fibroblast growth factor (aFGF) have been isolated. Nucleotide sequence analysis of these clones revealed that the coding region of the human aFGF gene is interrupted by two introns, located at precisely homologous locations to introns in four other members of the FGF gene family, strongly indicating a common evolutionary origin for these genes. Northern blot analyses of the multiple aFGF transcripts found in serum-stimulated human foreskin fibroblasts indicated that the aFGF gene also contains a third intron, lying in the 5' untranslated region.

Amino Acid Sequence↗
PubMed1988

Addition of growth hormone secretion signal to basic fibroblast growth factor results in cell transformation and secretion of aberrant forms of the protein.

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types. Unlike most growth factors, the primary translation product for bFGF appears to lack a secretory signal peptide. To explore the normal mode of bFGF release, as well as to investigate the growth factor's oncogenic potential, expression vectors were created for a bFGF cDNA and for a chimeric molecule in which the bFGF coding sequence was linked to the human growth hormone signal peptide sequence. Transfection of NIH3T3 cells with the bFGF cDNA vectors caused the synthesis of high levels of biologically active, cell-associated bFGF, but no evidence of transformation was detected. In contrast, the chimeric bFGF-signal peptide expression vector induced foci of transformation at a very high frequency. The transformed cells grew in soft agar and were tumorigenic in nude mice. The majority of the immunoreactive bFGF species made by the transformed cells was found in the conditioned medium and appeared to be posttranslationally modified, indicating that the chimeric bFGF-signal peptide molecule was processed through the secretory pathway. The secreted bFGF exhibited little mitogenic activity, suggesting that interaction of bFGF with its receptor likely occurs while the fusion protein is being processed along the secretory pathway.

Animals↗
PubMed1986

Plant glutamine synthetase complements a glnA mutation in Escherichia coli.

A glutamine synthetase gene from alfalfa (Medicago sativa) has been expressed in Escherichia coli after fusion of bacterial transcription and translation signals to a complete alfalfa glutamine synthetase coding sequence. Synthesis of the alfalfa glutamine synthetase enzyme in Escherichia coli was demonstrated by functional genetic complementation of a glutamine synthetase-deficient mutant and by immunoblotting analysis. These results should facilitate protein engineering and structure-function analysis of the plant enzyme.

Base Sequence↗
PubMed1984

Herbicide-resistant alfalfa cells: an example of gene amplification in plants.

We have selected alfalfa suspension cell lines that are 20- to 100-fold more resistant than wild-type cells to the nonselective herbicide L-phosphinothricin, a mixed competitive inhibitor of glutamine synthetase (GS). GS enzyme levels are three- to sevenfold elevated in the variant cell line. Partial amino acid sequences of two cyanogen bromide cleavage peptides have been determined on GS purified from the variant cell line and used to confirm the identity of a 276-base pair GS cDNA clone. Southern blot analysis of wild-type and variant cell DNAs using this cDNA as a probe indicate that resistance to L-phosphinothricin is a consequence of a four- to 11-fold amplification of one GS gene resulting in about an eightfold increase in mRNA levels, and an increased enzyme synthesis sufficient to overcome the toxic effects of the inhibitor.

Aminobutyrates↗
PubMed1983

[Efficiency of current technics for the isolation of viruses in water].

In an attempt to evaluate the efficiency of the current methodology for the concentration of viruses from water and wastewater, we have determined the viral concentration in raw sewage using three procedures: a) inoculation of cell cultures directly with the filtered sewage, b) concentration of the viruses from the sewage using nitrocellulose filters at a pH of 3.5 in the presence of AlCl3, followed by elution with Nutrient Broth and inoculation of the cell cultures with the eluant and c) flocculation of the Nutrient Broth after the elution as in b, dissolution of the precipitate in cell medium and inoculation of the cell cultures with this solution. The results show that this concentration procedure with nitrocellulose filters as recommended in the "Standard Methods for the Examination of Water and Wastewater" has an efficiency of 33% if compared with the direct inoculation. In a separate study, we have looked at the properties of electropositive Filters to bind polioviruses Typ I, Strain Lsc 2ab at neutral pH in the absence of salt. These filters, which could be used several times, were able to retain more then 99.5% of the viruses. However, they bound the viruses stronger that nitrocellulose filters and the elution with Nutrient Broth was never complete. After the problems with the elution are solved, these filters promise to simplify substantially the procedure for concentrating viruses from water.

Collodion↗
PubMed1980

Nucleotide sequence of an avian sarcoma virus oncogene (src) and proposed amino acid sequence for gene product.

The transforming gene (src) of avian sarcoma virus (ASV) and adjacent regions of the viral genome have been isolated by molelcular cloning of viral DNA. Their nucleotide sequence encompasses the whole of src and the portion of the gene env that encodes gp 37, one of two glycoproteins found in the viral envelope. Src encodes a single, hydrophobic protein with structural features that conform to previous descriptions descriptions of the gene product (pp60src). It appears that a single viral protein is responsible for both the initiation and maintenance of neoplastic transformation by avian sarcoma virus. Neither src nor its product bear any obvious structural relationship to several other viral oncogenes and their encoded proteins. Src is flanked by a repeated nucleotide sequence that may facilitate frequent deletion of the gene from the viral genome.

Alpharetrovirus↗
PubMed1980

The nucleotide sequence of an untranslated but conserved domain at the 3' end of the avian sarcoma virus genome.

The genomes of numerous avian retroviruses contain at their 3' termini a conserved domain denoted "c". The precise boundaries and function of "c" have been enigmas. In an effort to resolve these issues, we determined the sequence of over 900 nucleotides at the 3' end of the genome of the Schmidt-Ruppin subgroup A strain of avian sarcoma virus (ASV). We obtained the sequence from a suitable fragment of ASV DNA that had cloned into the single-stranded DNA phage M13mp2. Computer-assisted analysis of the sequence revealed the following structural features: i) the length of "c" - 473 nucleotides; ii) the 3' terminal domain of src, ending in an amber codon at the 5'boundary of "c"; iii) terminator codons that preclude continuous translation from "c"; iv) suitably located sequences that may serve as signals for the initiation of viral RNA synthesis and for the processing and/or polyadenylation of viral mRNA; v) a repeated sequence that flanks src and that could facilitate deletion of this gene; vi) repeated sequences within "c"; and vii) unexplained homologies between sequences in "c" and sequences in several other nucleic acids, including the 5' terminal domain of the ASV genome, tRNATrp and its inversion, the complement of tRNATrp and its inversion, and the 18S RNA of eukaryotic ribosomes. We conclude that "c" probably does not encode a protein, but its sequence may nevertheless serve several essential functions in viral replication.

Alpharetrovirus↗
PubMed1980

Sequence of the human insulin gene.

The human insulin gene contains two intervening sequences, one is within the region transcribed into the 5'-untranslated segment of the mRNA and the other interrupts the C-peptide encoding region. A comparison of the human with the rat insulin genes indicates potential regulatory regions in the DNA segment preceding the gene and suggests that the ancestral form of the insulin gene had two intervening sequences.

Alleles↗
PubMed1979

Isolation and characterization of a cloned rat insulin gene.

The two nonallelic genes, insulin I and II of the rat, are separated by at least 7 kb of DNA. There is no obvious similarity in the sequence organization surrounding each gene, although the coding regions of the genes themselves share extensive homology. In three strains of rat, the insulin II gene lies predominantly on a 4.0 kb Eco RI restriction fragment, whereas the insulin I gene is located on a 9.4 kb Eco RI fragment in the Hooded strain, on a 7.2 kb Eco RI fragment in the Osborne-Mendel strain, and on both a 9.4 and a 7.2 kb Eco RI fragment in Sprague-Dawley rats. The 9.4 kb Eco RI fragment from Hooded rat DNA was isolated using the lambda cloning system, and the nucleotide sequence of this isolated rat insulin I gene and adjacent regions was determined. A translation in one frame of the sequence of the cloned gene confirms the protein sequence determined for rat pre-proinsulin I. The coding region of this gene lacks intervening sequences, although a presumptive intervening sequence of 119 bp is located in the 5 untranslated region preceding the prehormone sequence. The junctions around the 119 bp segment are identical to those which flank intervening sequences of other eucaryotic genes, AGGT. The site of polyadenylation was determined by direct sequence comparison with rat insulin cDNA clones, and a potential 5 "capping" site is proposed. A DNA sequence preceding this 5 "capping" site in the rat insulin I gene, TATAAAGC, is homologous to corresponding regions in other eucaryotic genes that have been proposed as putative promoter sites for the initiation of transcription.

Animals↗
PubMed1977

Rat insulin genes: construction of plasmids containing the coding sequences.

Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.

Animals↗