The effect of azalomycin F on Ca2+ homeostasis in Trichoderma viride and Saccharomyces cerevisiae.

Azalomycin F (AMF), a macrocyclic lactone antibiotic, in concentrations of 10(-5) g/ml (10(-6) - 10(-5) mol/l) was found to stimulate both the 45Ca2+ influx and efflux in intact Trichoderma viride submerged mycelium and in cells of Saccharomyces cerevisiae without having Ca2+ ionophoric properties. AMF also inhibited ATP-dependent Ca2+ uptake in membrane fractions prepared from T. viride submerged mycelium. 45Ca2+ which had been accumulated in membrane fractions in an ATP-dependent manner was released upon addition of AMF. This release was observed in light organellar fractions (LOF) of S. cerevisiae and of T. viride submerged mycelium and, to a small extent, in heavy organellar fraction (HOF) of S. cerevisiae. No Ca2+ releasing effect of AMF was observed in HOF from T. viride submerged mycelium. In S. cerevisiae expressing Ca2+-dependent photoprotein aequorin, AMF induced transients of luminescence which reflect changes in the cytoplasmic Ca2+ concentration. The results suggest that the stimulation by AMF of the Ca2+ efflux from the mycelium (cells) could be explained by an increase of the cytoplasmic Ca2+ concentration due to the release of Ca2+ from microsomal membranes or to the stimulation of Ca2+ influx.

Ca2+ signal is generated only once in the mating pheromone response pathway in Saccharomyces cerevisiae.

The mating pheromone, alpha-factor, of the yeast Saccharomyces cerevisiae binds to the heterotrimeric G protein-coupled cell surface receptor of MATa cells and induces cellular responses necessary for mating. In higher eukaryotic cells, many hormones and growth factors rapidly mobilize a second messenger, Ca2+, by means of receptor-G protein signaling. Although striking similarities between the mechanisms of the receptor-G protein signaling in yeast and higher eukaryotes have long been known, it is still uncertain whether the pheromone rapidly mobilizes Ca2+ necessary for early events of the pheromone response. Here we reexamine this problem using sensitive methods for detecting Ca2+ fluxes and mobilization, and find no evidence that there is rapid Ca2+ influx leading to a rapid increase in the cytosolic free Ca2+ concentration. In addition, the yeast PLC1 deletion mutant lacking phosphoinositide-specific phospholipase C, a key enzyme for generating Ca2+ signals in higher eukaryotic cells, responds normally to the pheromone. These findings suggest that the receptor-G protein signaling does not utilize Ca2+ as a second messenger in the early stage of the pheromone response pathway. Since the receptor-G protein signaling does stimulate Ca2+ influx after early events have finished and this stimulation is essential for late events in the pheromone response pathway [Iida et al., (1990) J. Biol. Chem., 265: 13391-13399] Ca2+ may be used only once in the signal transduction pathway in unicellular eukaryotes such as yeast.

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence.

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris. HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (lambdamax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

Salicylic acid induces a cytosolic Ca2+ elevation in yeast.

Cytosolic free calcium ion concentration ([Ca2+]cyt) after a salicylic acid (SA)-stimulus was monitored in cells of the yeast Saccharomyces cerevisiae expressing apoaequorin, which constitutes a Ca(2+)-sensitive luminescent protein, aequorin, when combined with coelenterazine. SA induced a transient [Ca2+]cyt elevation that was dependent on the concentration of SA and pH of the SA solution. The SA-induced [Ca2+]cyt elevation was not reduced in Ca(2+)-deficient medium, suggesting that Ca2+ was mobilized from an intracellular Ca2+ store(s). Benzoic acid, butyric acid and sorbic acid did not induced a [Ca2+]cyt elevation.

Tracing the in vivo distribution and dynamics of cancer cells in mice by luminescence of aequorin- expressing transformants.

In order to trace the in vivo dynamics of cancer cells, we introduced jellyfish aequorin cDNA into metastatic (A-11) and nonmetastatic (3LL) Lewis lung cancer cell lines. The transformants (e.g., A-11-4 and 3LL-24) remained highly tumorigenic when they were s.c. injected into C57BL/6 mice and the A-11-4 clone produced metastatic tumors in the lung. Luminescence produced by the expressed aequorin was detectable in these transformants in culture as well as in cells of primary or secondary tumors. The limit of detection of the aequorin assay was approximately 90 cells, whereas approximately 5 x 10(4) cells could be macroscopically seen as metastatic foci in the lung. Thus, the aequorin assay is approximately 500-fold more sensitive than macroscopic examination in detecting cancer metastasis.

A bacterial cloning vector using a mutated Aequorea green fluorescent protein as an indicator.

The bacterial cloning vector, pGreenscript A, derived from the mutated Aequorea green fluorescent protein (GFP-S65A) gene, when expressed in E. coli produced colonies that showed yellow color under daylight and strong green fluorescence under long-wave ultraviolet light. The vector was used to select for inserted foreign genes based on the loss of the yellow color/green fluorescence of E. coli cells caused by the insertional inactivation of GFP production.

Mutagenesis of firefly luciferase shows that cysteine residues are not required for bioluminescence activity.

Conflicting reports exist in the literature as to whether sulfhydryl groups are essential for firefly luciferase activity. Inactivation of Photinus pyralis luciferase with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and site-directed mutagenesis demonstrate that the cysteine residues are not absolutely required for activity. However, loss of P. pyralis luciferase activity is observed when any of the 4 cysteine residues is replaced with serine.

Chemical nature of the light emitter of the Aequorea green fluorescent protein.

The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence (lambda max = 508 nm) when irradiated with long ultraviolet light. The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which fluorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission.

Regeneration and luminescence of aequorin in Chinese hamster ovary cells transformed with cDNA for apoaequorin.

Aequorin, a photoprotein which is regenerated from apoaequorin by incubation with coelenterazine, emits light when it binds Ca2+. The aim of this study was to determine if apoaequorin could be used in adherent mammalian cells for measuring cytosolic Ca2+, and imaging Ca2+, at the single cell level. Chinese hamster ovary (CHO-K1) cells were stably transformed with apoaequorin cDNA and expressed apoaequorin while attached to the culture dishes. Maximal luminescence intensity was obtained when 0.5 x 10(6) cells/ml were grown and incubated with 2.5 microM coelenterazine for 4 hr at 20 degrees C. Ca2+ mobilizing agents (ionomycin and maitotoxin) induced luminescence in CHO-K1 transformed cells. However, imaging of light emission from single cells proved to be unsuccessful. Ca2+ could be readily measured in the adherent CHO-K1 cells, but imaging was not possible at the single cell level.

Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells.

The localization, trafficking, and fluorescence of Aequorea green fluorescent protein (GFP) in cultured vertebrate cells transiently transfected with GFP cDNA were studied. Fluorescence of GFP in UV light was found to be strongest when cells were incubated at 30 degrees C but was barely visible at an incubation temperature of 37 degrees C. COS-1 cells, primary chicken embryonic retina cells, and carp epithelial cells were fluorescently labeled under these conditions. GFP was distributed uniformly throughout the cytoplasm and nucleus independent of cell type examined. When GFP was fused to PML protooncogene product, fluorescence was detected in a unique nuclear organelle pattern indistinguishable from that of PML protein, showing the potential use of GFP as a fluorescent tag. To analyze both function and intracellular trafficking of proteins fused to GFP, a GFP-human glucocorticoid receptor fusion construct was prepared. The GFP-human glucocorticoid receptor efficiently transactivated the mouse mammary tumor virus promoter in response to dexamethasone at 30 degrees C but not at 37 degrees C, indicating that temperature is important, even for function of the GFP fusion protein. The dexamethasone-induced translocation of GFP-human glucocorticoid receptor from cytoplasm to nucleus was complete within 15 min; the translocation could be monitored in a single living cell in real time.

Molecular evolution of the Ca(2+)-binding photoproteins of the Hydrozoa.

Alignment of the primary structures of the hydrozoan photoproteins, aequorin, mitrocomin, clytin and obelin showed very strong amino acid sequence identities. The Ca(2+)-binding sites of the proteins were found to be highly conserved. The Ca(2+)-binding sites were also homologous to the Ca(2+)-binding sites of other Ca(2+)-binding proteins. However, aequorin, mitrocomin, clytin and obelin differed from other Ca(2+)-binding proteins in that they contained a relatively large number of cysteine, tryptophan, histidine, proline and tyrosine residues, suggesting that these residues may have evolved as part of the light-emitting mechanism. Construction of a phylogenetic tree showed that aequorin, mitrocomin, clytin and obelin form a closely related group of proteins.

Real time imaging of transcriptional activity in live mouse preimplantation embryos using a secreted luciferase.

Study of gene expression kinetics during preimplantation mammalian development is difficult because of the limited amount of material and the usually destructive, static nature of molecular analyses. We describe continuous, noninvasive monitoring of gene expression in preimplantation embryos by using a secreted luminescent reporter, Vargula luciferase. Transgene expression profiles were followed by assaying aliquots of culture medium or by direct visualization of Vargula luciferase secretion from living embryos in real time through photon imaging. With this approach, it is possible to observe epigenetic modulations of gene expression and to link this over time to the developmental capacity of individual embryos. In addition, by developing a strategy where expression from integrated transgenes is enhanced relative to that from nonintegrated DNA, we provide evidence that rapid detection of transgene integration prior to the blastocyst stage should be possible. Thus, imaging of Vargula luciferase secretion may also be useful in the early screening of embryos, for example, in the production of transgenic livestock.

Evidence for redox forms of the Aequorea green fluorescent protein.

Highly purified recombinant Aequorea green fluorescent protein is able to undergo a reversible oxidation-reduction reaction in the presence of molecular oxygen. In the oxidized form in near UV light, the protein is highly fluorescent, but when reduced with sodium dithionite, it becomes completely non-fluorescent. On exposure to molecular oxygen the reduced, non-fluorescent protein reverts to its original fluorescent state.

Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein.

Expression of the cDNA for Aequorea green fluorescent protein in E. coli yielded a fused protein with fluorescence excitation and emission spectra virtually identical to those of the native green fluorescent protein. Further, a solution of the protein, when mixed with aequorin and calcium ion, emitted a greenish luminescence characteristic of the in vivo luminescence of the animal, indicating a radiationless energy transfer to the protein.

Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA.

A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)i], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)i] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells at all stages of development exhibit a sharp transient increase in [(Ca2+)i] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)i] and show three times as large a response to cAMP stimulation as prespore cells.

Cloning, expression and sequence analysis of cDNA for the Ca(2+)-binding photoprotein, mitrocomin.

The primary structure of mitrocomin consists of 190 amino acid residues, with three Ca(2+)-binding sites and a tyrosine residue at the C-terminus. Mitrocomin shows an amino acid sequence homology of 67.9% and 60.7% when compared with aequorin and clytin, respectively. The amino acid residues Cys152, His58, His169, Trp12, Trp86, Trp108, Trp129 and Trp173 are conserved in all three photoproteins, suggesting that they play a role in light emission.

Mass spectrometric evidence for a disulfide bond in aequorin regeneration.

Tryptic digests of purified recombinant apoaequorin were analyzed, before and after reduction with DTT, by fast atom bombardment mass spectrometry. The results showed that apoaequorin contains a disulfide bond between Cys145 and Cys152 and that the reduction of this bond is involved in the regeneration of aequorin.

Bioluminescence of the Ca(2+)-binding photoprotein, aequorin, after histidine modification.

Modification studies of the 5 histidine residues in aequorin employing site-directed mutagenesis and diethyl pyrocarbonate suggested that His169 may be the site of binding of molecular oxygen in aequorin. The modification of this residue led to complete loss of activity, whereas modification of the remaining 4 histidine residues yielded mutant aequorins with varying bioluminescence activities.

Cloning and sequence analysis of cDNA for the Ca(2+)-activated photoprotein, clytin.

Clytin is a member of the aequorin family of photoproteins. It is made up of 189 amino acid residues, contains 3 Ca(2+)-binding sites, and shows 62% homology in amino acid residues to those in aequorin. The cysteine, tryptophan, and histidine residues, and the C-terminal proline, that are conserved in aequorin and clytin may be involved in the Ca(2+)-activated bioluminescence of the two proteins. Clytin may also prove useful in the determination of Ca2+.