A yeast genetic assay for caspase cleavage of the amyloid-beta precursor protein.

A functional assay for proteolytic processing of the amyloid precursor protein (APP) was set up in yeast. This consisted of a membrane-bound chimeric protein containing the beta-secretase cleaved C-terminal fragment of APP fused to the Ga14 transcription factor. Using this chimera in a GAL-reporter yeast strain, an expression library of human cDNAs was screened for clones that could activate the GAL-reporter genes by proteolytic processing of the membrane-bound APP-Gal4. Two human proteases, caspase-3 and caspase-8, were identified and confirmed to act by a mechanism that involved proteolysis at the site in the APP-Gal4 chimera that corresponded to the natural caspase cleavage site in APP, thus linking a readily scorable phenotype to proteolytic processing of APP. The activation of caspase-3 involved a mechanism that was independent of aspartic acid residue 175 at the cleavage site normally required for processing of caspase-3.

Saccharomyces cerevisiae is capable of de Novo pantothenic acid biosynthesis involving a novel pathway of beta-alanine production from spermine.

Pantothenic acid and beta-alanine are metabolic intermediates in coenzyme A biosynthesis. Using a functional screen in the yeast Saccharomyces cerevisiae, a putative amine oxidase, encoded by FMS1, was found to be rate-limiting for beta-alanine and pantothenic acid biosynthesis. Overexpression of FMS1 caused excess pantothenic acid to be excreted into the medium, whereas deletion mutants required beta-alanine or pantothenic acid for growth. Furthermore, yeast genes ECM31 and YIL145c, which both have structural homology to genes of the bacterial pantothenic acid pathway, were also required for pantothenic acid biosynthesis. The homology of FMS1 to FAD-containing amine oxidases and its role in beta-alanine biosynthesis suggested that its substrates are polyamines. Indeed, we found that all the enzymes of the polyamine pathway in yeast are necessary for beta-alanine biosynthesis; spe1Delta, spe2Delta, spe3Delta, and spe4Delta are all beta-alanine auxotrophs. Thus, contrary to previous reports, yeast is naturally capable of pantothenic acid biosynthesis, and the beta-alanine is derived from methionine via a pathway involving spermine. These findings should facilitate the identification of further enzymes and biochemical pathways involved in polyamine degradation and pantothenic acid biosynthesis in S. cerevisiae and raise questions about these pathways in other organisms.

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.

A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance.

The ability to counterselect, as well as to select for, a genetic marker has numerous applications in microbial genetics. Described here is the use of 5-fluoroanthranilic acid for the counterselection of TRP1, a commonly used genetic marker in the yeast Saccharomyces cerevisiae. Counterselection using 5-fluoroanthranilic acid involves antimetabolism by the enzymes of the tryptophan biosynthetic pathway, such that trp1, trp3, trp4 or trp5 strains, which lack enzymes required for the conversion of anthranilic acid to tryptophan, are resistant to 5-fluoroanthranilic acid. Commonly used genetic procedures, such as selection for loss of a chromosomally integrated plasmid, and a replica-plating method to rapidly assess genetic linkage in self-replicating shuttle vectors, can now be carried out using the TRP1 marker gene. In addition, novel tryptophan auxotrophs can be selected using 5-fluoroanthranilic acid.

Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis.

Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.

Identification and concerted function of two receptor binding surfaces on basic fibroblast growth factor required for mitogenesis.

Members of the fibroblast growth factor (FGF) family promote angiogenesis and wound repair, modulate early developmental events and survival of neurons, and have been associated with the pathogenesis of various diseases. FGFs interact with specific FGF receptors (FGFRs) and heparan sulfate proteoglycans on cell surfaces to mediate mitogenesis. Using protein structure-based site-directed mutagenesis of basic FGF (bFGF), we have identified two FGFR binding sites on bFGF which act in concert to initiate signal transduction. Both FGFR binding surfaces are distinct from the heparan sulfate proteoglycan binding domain. The primary, higher affinity, binding interaction comprises a cluster of solvent exposed hydrophobic amino acids (Tyr-24, Tyr-103, Leu-140, and Met-142), and two polar residues (Arg-44 and Asn-101). The hydrophobic contacts dominate the primary binding interaction and provide approximately 75% of the binding affinity. The secondary FGFR binding site on bFGF has an approximately 250-fold lower affinity and is composed of amino acids Lys-110, Tyr-111, and Trp-114 in a surface-exposed type I beta-turn (formerly known as the putative receptor binding loop). Binding of FGFR to both bFGF surfaces in a stoichiometry of 2FGFR:1bFGF is required for growth factor mediated cell proliferation. This represents a mechanism for the fibroblast growth factor/receptor family in which FGF facilitates FGFR dimerization and subsequent signal transduction events as a monomeric ligand.

Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli. Cleavage of a fusion protein with cyanogen bromide.

Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8----Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine. The enzyme has low activity on monomeric 1,2-diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site.