Edmund Tischer

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.

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

The formation of β-amyloid by processing of its precursor protein is a characteristic of Alzheimers disease. Two proteolytic cleavages produce the amino and carboxyl termini of β-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 β-amyloid domain. Substitution of negatively charged residues across positions 40-46 of the β-amyloid domain precluded both β-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 β-amyloid, possibly without membrane destruction. Deletions within the carboxyl-terminal domain, including 4 residues spanning positions 39-42 of β-amyloid, resulted in formation of the β-amyloid peptide. Substituting residues 38-47 or 39-56 of the β-amyloid domain in the precursor with a transmembrane sequence from another protein yielded a ∼4-kDa β-amyloid peptide, reflecting a loose residue specificity for carboxyl-terminal processing to β-amyloid.

CLONING OF DROSOPHILA MELANOGASTER ECORI DNA FRAGMENTS

ABSTRACT Recombinant plasmid DNAs were constructed in. vitro by ligating Eco RI endonuclease-cleaved Escherichia coli plasmids pSF2124 or pMB9 and selected large fragments of EcoR I endonuclease digested Prosophila melanogaster DNA. The mixture of ligated DNAs was used to transform E. coli K12 strain RRl. Transformed cells were selected for ampicillin resistance (pSF2124) or for tetracycline resistance (pMB9) and were further analyzed for the presence of Prosophila DNA. Fourteen clones containing recombinant plasmid DNAs were studied. Hybridization experiments with most of the plasmid DNAs yielded Cot½ values consistent with a unique sequence of that molecular weight with no internal repetition of base sequences. Only three plasmids contained DNA sequences repeated between 10 and 100-fold in the Drosophila genome.