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Methods (San Diego, Calif.) Dec 2012A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are...
A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light/cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems.
Topics: Arabidopsis Proteins; Basic Helix-Loop-Helix Transcription Factors; Cryptochromes; DNA-Binding Proteins; Galactokinase; Gene Expression Regulation, Fungal; Genes, Reporter; Light; Phycobilins; Phycocyanin; Phytochrome B; Promoter Regions, Genetic; Protein Binding; Protein Interaction Mapping; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors; Transcription, Genetic; Two-Hybrid System Techniques; beta-Galactosidase
PubMed: 22922268
DOI: 10.1016/j.ymeth.2012.08.004 -
Nature Communications May 2018The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned...
The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. Here, we present L-SCRaMbLE, a light-controlled Cre recombinase for use in the yeast Saccharomyces cerevisiae. L-SCRaMbLE allows tight regulation of recombinase activity with up to 179-fold induction upon exposure to red light. The extent of recombination depends on induction time and concentration of the chromophore phycocyanobilin (PCB), which can be easily adjusted. The tool presented here provides improved recombination control over the previously reported estradiol-dependent SCRaMbLE induction system, mediating a larger variety of possible recombination events in SCRaMbLE-ing a reporter plasmid. Thereby, L-SCRaMbLE boosts the potential for further customization and provides a facile application for use in the S. cerevisiae genome re-engineering project Sc2.0 or in other recombination-based systems.
Topics: Clone Cells; Gene Editing; Gene Expression; Genes, Synthetic; Genetic Engineering; Genome, Fungal; Integrases; Light; Phycobilins; Phycocyanin; Plasmids; Recombination, Genetic; Saccharomyces cerevisiae; Selection, Genetic
PubMed: 29789561
DOI: 10.1038/s41467-017-02208-6 -
European Journal of Biochemistry Feb 1969
Topics: Alkalies; Bile Acids and Salts; Chemical Phenomena; Chemistry; Chromatography, Thin Layer; Esters; Eukaryota; Imides; Methods; Models, Structural; Oxidation-Reduction; Peptides; Photosynthesis; Pigments, Biological; Plant Proteins; Spectrophotometry
PubMed: 5776242
DOI: 10.1111/j.1432-1033.1969.tb19637.x -
Controllable Phycobilin Modification: An Alternative Photoacclimation Response in Cryptophyte Algae.ACS Central Science Mar 2022Cryptophyte algae are well-known for their ability to survive under low light conditions using their auxiliary light harvesting antennas, phycobiliproteins. Mainly...
Cryptophyte algae are well-known for their ability to survive under low light conditions using their auxiliary light harvesting antennas, phycobiliproteins. Mainly acting to absorb light where chlorophyll cannot (500-650 nm), phycobiliproteins also play an instrumental role in helping cryptophyte algae respond to changes in light intensity through the process of photoacclimation. Until recently, photoacclimation in cryptophyte algae was only observed as a change in the cellular concentration of phycobiliproteins; however, an additional photoacclimation response was recently discovered that causes shifts in the phycobiliprotein absorbance peaks following growth under red, blue, or green light. Here, we reproduce this newly identified photoacclimation response in two species of cryptophyte algae and elucidate the origin of the response on the protein level. We compare isolated native and photoacclimated phycobiliproteins for these two species using spectroscopy and mass spectrometry, and we report the X-ray structures of each phycobiliprotein and the corresponding photoacclimated complex. We find that neither the protein sequences nor the protein structures are modified by photoacclimation. We conclude that cryptophyte algae change one chromophore in the phycobiliprotein β subunits in response to changes in the spectral quality of light. Ultrafast pump-probe spectroscopy shows that the energy transfer is weakly affected by photoacclimation.
PubMed: 35350600
DOI: 10.1021/acscentsci.1c01209 -
Current Neuropharmacology 2021The edible cyanobacterium Spirulina platensis and its chief biliprotein C-Phycocyanin have shown protective activity in animal models of diverse human health diseases,...
The edible cyanobacterium Spirulina platensis and its chief biliprotein C-Phycocyanin have shown protective activity in animal models of diverse human health diseases, often reflecting antioxidant and anti-inflammatory effects. The beneficial effects of C-Phycocyanin seem likely to be primarily attributable to its covalently attached chromophore Phycocyanobilin (PCB). Within cells, biliverdin is generated from free heme and it is subsequently reduced to bilirubin. Although bilirubin can function as an oxidant scavenger, its potent antioxidant activity reflects its ability to inactivate some isoforms of NADPH oxidase. Free bilirubin can also function as an agonist for the aryl hydrocarbon receptor (AhR); this may explain its ability to promote protective Treg activity in cellular and rodent models of inflammatory disease. AhR agonists also promote transcription of the gene coding for Nrf-2, and hence can up-regulate phase 2 induction of antioxidant enzymes, such as HO-1. Hence, it is proposed that C-Phycocyanin/PCB chiefly exert their protective effects via inhibition of NADPH oxidase activity, as well as by AhR agonism that both induces Treg activity and up-regulates phase 2 induction. This simple model may explain their potent antioxidant/antiinflammatory effects. Additionally, PCB might mimic biliverdin in activating anti-inflammatory signaling mediated by biliverdin reductase. This essay reviews recent research in which CPhycocyanin and/or PCB, administered orally, parenterally, or intranasally, have achieved marked protective effects in rodent and cell culture models of Ischemic Stroke and Multiple Sclerosis, and suggests that these agents may likewise be protective for Alzheimer's disease, Parkinson's disease, and in COVID-19 and its neurological complications.
Topics: Animals; COVID-19; Dietary Supplements; Humans; Neurodegenerative Diseases; Phycobilins; Phycocyanin; SARS-CoV-2
PubMed: 33829974
DOI: 10.2174/1570159X19666210408123807 -
Proceedings of the National Academy of... Feb 2013The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a...
The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period.
Topics: Bile Pigments; Biliverdine; Biocatalysis; Chlamydomonas reinhardtii; Chlorophyll; Chloroplasts; Gene Expression Profiling; Gene Expression Regulation, Plant; Gene Regulatory Networks; Genes, Plant; Heme; Heme Oxygenase (Decyclizing); Iron; Light; Mutation; Oxidoreductases; Phenotype; Phototrophic Processes; Phycobilins; Phycocyanin; Pigmentation; Plants, Genetically Modified; Signal Transduction; Subcellular Fractions
PubMed: 23345435
DOI: 10.1073/pnas.1222375110 -
American Journal of Physiology.... Jan 2013We and other investigators have reported that bilirubin and its precursor biliverdin may have beneficial effects on diabetic vascular complications, including...
We and other investigators have reported that bilirubin and its precursor biliverdin may have beneficial effects on diabetic vascular complications, including nephropathy, via its antioxidant effects. Here, we investigated whether phycocyanin derived from Spirulina platensis, a blue-green algae, and its chromophore phycocyanobilin, which has a chemical structure similar to that of biliverdin, protect against oxidative stress and renal dysfunction in db/db mice, a rodent model for Type 2 diabetes. Oral administration of phycocyanin (300 mg/kg) for 10 wk protected against albuminuria and renal mesangial expansion in db/db mice, and normalized tumor growth factor-β and fibronectin expression. Phycocyanin also normalized urinary and renal oxidative stress markers and the expression of NAD(P)H oxidase components. Similar antioxidant effects were observed following oral administration of phycocyanobilin (15 mg/kg) for 2 wk. Phycocyanobilin, bilirubin, and biliverdin also inhibited NADPH dependent superoxide production in cultured renal mesangial cells. In conclusion, oral administration of phycocyanin and phycocyanobilin may offer a novel and feasible therapeutic approach for preventing diabetic nephropathy.
Topics: Administration, Oral; Albuminuria; Animals; Antioxidants; Bilirubin; Biliverdine; Cells, Cultured; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Fibronectins; Gene Expression Regulation; Humans; Kidney; Male; Mice; Mice, Inbred C57BL; NADPH Oxidases; Oxidative Stress; Phycobilins; Phycocyanin; Spirulina; Superoxides; Time Factors; Transforming Growth Factor beta
PubMed: 23115122
DOI: 10.1152/ajpregu.00648.2011 -
Proceedings of the National Academy of... Jan 2020A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and...
A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on F - F difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.
Topics: Adenylyl Cyclases; Crystallography; Crystallography, X-Ray; Cyanobacteria; Cyclic GMP; Light; Models, Molecular; Phosphoric Diester Hydrolases; Photoreceptor Cells; Phycobilins; Phycocyanin; Phytochrome; Protein Conformation; Protein Domains; Thermosynechococcus; Trans-Activators
PubMed: 31852825
DOI: 10.1073/pnas.1912041116 -
The Journal of General Physiology Nov 1959Quantum yield measurements were made with the red alga Porphyridium cruentum, cultured so as to give different proportions of chlorophyll and phycobilins. Totally...
Quantum yield measurements were made with the red alga Porphyridium cruentum, cultured so as to give different proportions of chlorophyll and phycobilins. Totally absorbing suspensions were used so that there was no uncertainty in the amount of energy absorbed. These measurements have shown that chlorophyll, in this alga, has a photosynthetic efficiency as high as in other algal groups, and higher than the phycobilins-at least at wave lengths shorter than about 650 mmicro. Wave lengths longer than this are beyond the range of maximum efficiency of chlorophyll. Under specified conditions of temperature and supplementary light full efficiency may be extended to longer wave lengths. The results of these measurements have made it unnecessary to suppose that in red algae chlorophyll plays a minor role while the phycobilins are the photosynthetic sensitizers of primary importance.
Topics: Chlorophyll; Chlorophyll A; Light; Photosynthesis; Porphyridium; Rhodophyta; Temperature
PubMed: 13804675
DOI: 10.1085/jgp.43.2.251 -
Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling.Proceedings of the National Academy of... Nov 2017Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced...
Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)-phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB-PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB-PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB-PIF system, which paves the way for its application to a living animal.
Topics: Cell Line, Tumor; Ferredoxin-NADP Reductase; Ferredoxins; Genetic Vectors; HeLa Cells; Heme Oxygenase (Decyclizing); Humans; Light; Optogenetics; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Phycobilins; Phycocyanin; Signal Transduction
PubMed: 29078307
DOI: 10.1073/pnas.1707190114