Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins

Much effort has been dedicated to the design of significantly red shifted variants of the green fluorescent protein (GFP) from Aequoria victora (av). These approaches have been based on classical engineering with the 20 canonical amino acids. We report here an expansion of these efforts by incorporation of an amino substituted variant of tryptophan into the "cyan" GFP mutant, which turned it into a "gold" variant. This variant possesses a red shift in emission unprecedented for any avFP, similar to "red" FPs, but with enhanced stability and a very low aggregation tendency. An increasing number of non-natural amino acids are available for chromophore redesign (by engineering of the genetic code) and enable new general strategies to generate novel classes of tailor-made GFP proteins.     

How<Emphasis Type="Italic"> Daphnia</Emphasis> copes with excess carbon in its food

Animals that maintain near homeostatic elemental ratios may get rid of excess ingested elements from their food in different ways. C regulation was studied in juveniles of Daphnia magna feeding on two Selenastrum capricornutum cultures contrasting in P content (400 and 80 C:P atomic ratios). Both cultures were labelled with ¹⁴C in order to measure Daphnia ingestion and assimilation rates. No significant difference in ingestion rates was observed between P-low and P-rich food, whereas the net assimilation of ¹⁴C was higher in the treatment with P-rich algae. Some Daphnia were also homogeneously labelled over 5 days on radioactive algae to estimate respiration rates and excretion rates of dissolved organic C (DOC). The respiration rate for Daphnia fed with high C:P algae (38.7% of body C day^-1) was significantly higher than for those feeding on low C:P algae (25.3% of body C day^-1). The DOC excretion rate was also higher when animals were fed on P-low algae (13.4% of body C day^-1) than on P-rich algae (5.7% of body C day^-1) . When corrected for respiratory losses, total assimilation of C did not differ significantly between treatments (around 60% of body C day^-1). Judging from these experiments, D. magna can maintain its stoichiometric balance when feeding on unbalanced diets (high C:P) primarily by disposing of excess dietary C via respiration and excretion of DOC.     

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.     

Intracellular redistribution of protein kinase D2 in response to G-protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine protein kinases: PKC mu/PKD, PKD2, and PKC nu/PKD3. While PKD has been the focus of most studies to date, no information is available on the intracellular distribution of PKD2. Consequently, we examined the mechanism that regulates its intracellular distribution in human pancreatic carcinoma Panc-1 cells. Analysis of the intracellular steady-state distribution of fluorescent-tagged PKD2 in unstimulated cells indicated that this kinase is predominantly cytoplasmic. Cell stimulation with the G protein-coupled receptor agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD2 by a mechanism that requires PKC activity. In contrast to the other PKD isoenzymes, PKD2 activation did not induce its redistribution from the cytoplasm to the nucleus. Thus, this study demonstrates that the regulation of the distribution of PKD2 is distinct from other PKD isoenzymes, and suggests that the differential spatio-temporal localization of these signaling molecules regulates their specific signaling properties.     

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.     

Imazalil-cyclomaltoheptaose (beta-cyclodextrin) inclusion complex: preparation by supercritical carbon dioxide and 13C CPMAS and 1H NMR characterization

An inclusion complex between imazalil (IMZ), a selected fungicide, and cyclomaltoheptaose (beta-cyclodextrin, betaCD) was obtained using supercritical fluid carbon dioxide. The best preparation conditions were determined, and the inclusion complex was investigated by means of 1H NMR spectroscopy in aqueous solution and 13C CPMAS NMR spectroscopy in the solid state. Information on the geometry of the betaCD/IMZ complex was obtained from ROESY spectroscopy, while the dynamics of the inclusion complex in the kilohertz range was obtained from the proton spin-lattice relaxation times in the rotating frame, T(1rho) (1H).     

A microscope-based screening platform for large-scale functional protein analysis in intact cells

A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects.     

Selective feeding by larval dytiscids (Coleoptera:Dytiscidae) and effects of fish predation on upper littoral zone macroinvertebrate communities of arctic lakes

Carbon and nitrogen stable isotopic data from the primary producers in mangrove ecosystems are needed to investigate trophic links and biogeochemical cycling. Compared with other mangrove species (e.g. Rhizophora mangle) very few measurements have been conducted on the white mangrove, Laguncularia racemosa. The carbon and nitrogen stable isotopic and elemental compositions of L. racemosa were analyzed and compared from Florida and Belize. ¹³C values of L. racemosa from Florida (mean = –26.4) were slightly higher than those from Twin Cays, Belize (mean = -27.4), which may be due to higher salinity in some parts of the Florida site. There was no difference between the ¹⁵N values from L. racemosa from these two sites (Florida mean = 0.6; Belize mean = 0.3), which are indicative of nitrogen derived from nitrogen fixation in a planktonic marine system. However, higher ¹⁵N values from L. racemosa at Man of War Cay in Belize (11.4 and 12.3), which is fertilized by roosting marine birds (14.0), illustrate that L. racemosa can sensitively reflect alternative nitrogen sources. Although the isotopic data could not distinguish between Avicennia germinans, R. mangle and L. racemosa in Belize the L. racemosa had considerably higher C/N ratios (46.5 – 116.1) compared with the Florida samples (42.2 – 76.0) or the other mangrove species. Unlike some previous findings from R. mangle, substrate characteristics (e.g. salinity, NH₄ ⁺, and H₂S) were not related to the isotopic or elemental composition of L. racemosa. ¹³C, ¹⁵N and C/N were analyzed for ecosystem components from L. racemosa habitats at Twin Cays, including other plants (e.g. R. mangle, A. germinans and seagrass), detritus, microbial mats and sediments. Results from mass-balance calculations show that mangrove detritus composes very little of the sediment, which is principally composed of microbial biomass (80 – 90%). Detritus at some sites is also influenced by sources other than that from L. racemosa, including seagrass leaves.     

Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP⁺/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, K _m for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg²⁺ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots. Received: 21 June 2000 / Accepted: 28 July 2000     

Promoter strength and tissue specificity effects on growth of tomato plants transformed with maize sucrose-phosphate synthase

When sucrose-phosphate synthase (SPS; EC 2.4.1.14) is expressed in tomato (Lycopersicon esculentum Mill.) from a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit (rbcS) promoter, yields are often unchanged but when SPS is expressed from a Cauliflower Mosaic Virus 35S promoter, yield is enhanced up to 80%. Two explanations for this phenomenon are (i) that expression of SPS in tissues other than leaves accounts for the increased yield or (ii) that the lower level of expression directed by the 35S promoter is more beneficial than the high level of expression directed by the rbcS promoter. To test the first hypothesis, we conducted a reciprocal graft experiment, which showed that root SPS activity did not substantially affect growth. To test the second hypothesis, we conducted a field trial using a backcrossed, segregating, population of SPS-transformed plants derived from 35S and rbcS lines. The optimal dose of SPS activity for growth was approximately twice that of the wild type regardless of which promoter was used. The effect of SPS on growth was the result of a shift in partitioning of carbon among starch, sucrose, and ionic compounds (primarily amino acids), rather than of an increase in net photosynthesis. Excessive SPS activity resulted in a decreased rate of amino acid synthesis, which could explain the non-linear response of plant growth to the level of SPS expression. Received: 23 May 2000 / Accepted: 24 July 2000