Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage.

The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution.

An anion channel in Arabidopsis hypocotyls activated by blue light.

A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.

A new high molecular weight immunoglobulin class from the carcharhine shark: implications for the properties of the primordial immunoglobulin.

All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy-chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution.

Non-iron porphyrins cause tumbling to blue light by an Escherichia coli mutant defective in hemG.

Previously we showed that an Escherichia coli hemH mutant, defective in the ultimate step of heme synthesis, ferrochelatase, is somewhat better than 100-fold more sensitive than its wild-type parent in tumbling to blue light. Here we explore the effect of a hemG mutant, defective in the penultimate step, protoporphyrinogen oxidase. We found that a hemG mutant also is somewhat better than 100-fold more sensitive in tumbling to blue light compared to its wild-type parent. The amount of non-iron porphyrins accumulated in hemG or hemH mutants was more than 100-fold greater than in wild type. The nature of these accumulated porphyrins is described. When heme was present, as in the wild type, the non-iron (non-heme) porphyrins were maintained at a relatively low concentration and tumbling to blue light at an intensity effective for hemG or hemH did not occur. The function of tumbling to light is most likely to allow escape from the lethality of intense light.

Close correspondence between the action spectra for the blue light responses of the guard cell and coleoptile chloroplasts, and the spectra for blue light-dependent stomatal opening and coleoptile phototropism.

Fluorescence spectroscopy was used to characterize blue light responses from chloroplasts of adaxial guard cells from Pima cotton (Gossypium barbadense) and coleoptile tips from corn (Zea mays). The chloroplast response to blue light was quantified by measurements of the blue light-induced enhancement of a red light-stimulated quenching of chlorophyll a fluorescence. In adaxial (upper) guard cells, low fluence rates of blue light applied under saturating fluence rates of red light enhanced the red light-stimulated fluorescence quenching by up to 50%. In contrast, added blue light did not alter the red light-stimulated quenching from abaxial (lower) guard cells. This response pattern paralleled the blue light sensitivity of stomatal opening in the two leaf surfaces. An action spectrum for the blue light-induced enhancement of the red light-stimulated quenching showed a major peak at 450 nm and two minor peaks at 420 and 470 nm. This spectrum matched closely an action spectrum for blue light-stimulated stomatal opening. Coleoptile chloroplasts also showed an enhancement by blue light of red light-stimulated quenching. The action spectrum of this response, showing a major peak at 450 nm, a minor peak at 470 nm, and a shoulder at 430 nm, closely matched an action spectrum for blue light-stimulated coleoptile phototropism. Both action spectra match the absorption spectrum of zeaxanthin, a chloroplastic carotenoid recently implicated in blue light photoreception of both guard cells and coleoptiles. The remarkable similarity between the action spectra for the blue light responses of guard cells and coleoptile chloroplasts and the spectra for blue light-stimulated stomatal opening and phototropism, coupled to the recently reported evidence on a role of zeaxanthin in blue light photoreception, indicates that the guard cell and coleoptile chloroplasts specialize in sensory transduction.

Red/far-red and blue light-responsive regions of maize rbcS-m3 are active in bundle sheath and mesophyll cells, respectively.

Leaves of the C4 plant maize have two major types of photosynthetic cells: a ring of five large bundle sheath cells (BSC) surrounds each vascular bundle and smaller mesophyll cells (MC) lie between the cylinders of bundle sheath cells. The enzyme ribulose bisphosphate carboxylase/oxygenase is encoded by nuclear rbcS and chloroplast rbcL genes. It is not present in MC but is abundant in adjacent BSC of green leaves. As reported previously, the separate regions of rbcS-m3, which are required for stimulating transcription of the gene in BSC and for suppressing expression of reporter genes in MC, were identified by an in situ expression assay; expression was not suppressed in MC until after leaves of dark-grown seedlings had been illuminated for 24 h. Now we have found that transient expression of rbcS-m3 reporter genes is stimulated in BSC via a red/far-red reversible phytochrome photoperception and signal transduction system but that blue light is required for suppressing rbcS-m3 reporter gene expression in MC. Blue light is also required for the suppression system to develop in MC. Thus, the maize gene rbcS-m3 contains certain sequences that are responsive to a phytochrome photoperception and signal transduction system and other regions that respond to a UVA/blue light photoperception and signal transduction system. Various models of "coaction" of plant photoreceptors have been advanced; these observations show the basis for one type of coaction.

Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.

Phototaxis away from blue light by an Escherichia coli mutant accumulating protoporphyrin IX.

The hemH gene of Escherichia coli encodes ferrochelatase (EC 4.99.1.1), the enzyme that catalyzes the last step in the production of heme, namely the synthesis of heme from protoporphyrin IX plus Fe2+. The behavioral responses to light were studied in E. coli carrying a hemH mutation. It was shown that the hemH mutant displayed a tumbling response upon illumination and a running response upon removal of the light. The most effect light to induce a tumbling response in the hemH mutant was blue light (396-450 nm). The chemotaxis machinery was needed for the light-induced tumbling response in the hemH mutant. The bacterial defect is an analog of the human inherited disease erythropoietic protoporphyria.

A chimeric light-regulated amino acid transport system allows the isolation of blue light regulator (blr) mutants of Neurospora crassa.

We have developed a system for the isolation of Neurospora crassa mutants that shows altered responses to blue light. To this end we have used the light-regulated promoter of the albino-3 gene fused to the neutral amino acid permease gene mtr. The product of the mtr gene is required for the uptake of neutral aliphatic and aromatic amino acids, as well as toxic analogs such as p-flurophenylalanine or 4-methyltryptophan. mtr trp-2-carrying cells were transformed with the al-3 promoter-mtr wild-type gene (al-3p-mtr+) to obtain a strain with a light-regulated tryptophan uptake. This strain is sensitive to p-fluorophenylalanine when grown under illumination and resistant when grown in the dark. UV mutagenesis of the al-3p-mtr(+)-carrying strain allowed us to isolate two mutant strains, BLR-1 and BLR-2 (blue light regulator), that are light-resistant to p-fluorophenylalanine and have lost the ability to grow on tryptophan. These two strains have a pale-orange phenotype and show down-regulation of all the photoregulated genes tested (al-3, al-1, con-8, and con-10). Mutations in the BLR strains are not allelic with white collar 1 or white collar 2, regulatory genes that are also involved in the response to blue light.