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Lei, L, Xu A, Panin VM, Irvine KD.  2003.  An O-fucose site in the ligand binding domain inhibits Notch activation. Development (Cambridge, England). 130:6411-21. AbstractWebsite
Two glycosyltransferases that transfer sugars to EGF domains, OFUT1 and Fringe, regulate Notch signaling. However, sites of O-fucosylation on Notch that influence Notch activation have not been previously identified. Moreover, the influences of OFUT1 and Fringe on Notch activation can be positive or negative, depending on their levels of expression and on whether Delta or Serrate is signaling to Notch. Here, we describe the consequences of eliminating individual, highly conserved sites of O-fucose attachment to Notch. Our results indicate that glycosylation of an EGF domain proposed to be essential for ligand binding, EGF12, is crucial to the inhibition of Serrate-to-Notch signaling by Fringe. Expression of an EGF12 mutant of Notch (N-EGF12f) allows Notch activation by Serrate even in the presence of Fringe. By contrast, elimination of three other highly conserved sites of O-fucosylation does not have detectable effects. Binding assays with a soluble Notch extracellular domain fusion protein and ligand-expressing cells indicate that the NEGF12f mutation can influence Notch activation by preventing Fringe from blocking Notch-Serrate binding. The N-EGF12f mutant can substitute for endogenous Notch during embryonic neurogenesis, but not at the dorsoventral boundary of the wing. Thus, inhibition of Notch-Serrate binding by O-fucosylation of EGF12 might be needed in certain contexts to allow efficient Notch signaling.
Ruettinger, W, Yagi M, Wolf K, Bernasek S, Dismukes GC.  2000.  O2 Evolution from the Manganese−Oxo Cubane Core Mn4O46+:  A Molecular Mimic of the Photosynthetic Water Oxidation Enzyme? Journal of the American Chemical Society. 122:10353-10357. AbstractWebsite
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Mekler, V, Minakhin L, Severinov K.  2011.  On the role of downstream RNA polymerase-promoter interactions in formation of transcription initiation complex.. J. Biol. Chem.. 286:22600-22608.
Altman, S, Bassler BL, Beckwith J, Belfort M, Berg HC, Bloom B, Brenchley JE, Campbell A, Collier JR, Connell N et al..  2005.  An open letter to Elias Zerhouni.. Science (New York, N.Y.). 307(5714):1409-10.
Chakraborty, A, Wang D, Ebright YW, Korlann Y, Kortkhonjia E, Kim T, Chowdhury S, Wigneshweraraj S, Irschik H, Jansen R et al..  2012.  Opening and closing of the bacterial RNA polymerase clamp.. Science (New York, N.Y.). 337(6094):591-5. AbstractWebsite
Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.
Ananyev, GM, Carrieri D, Dismukes GC.  2008.  Optimization of metabolic capacity and flux through environmental cues to maximize hydrogen production by the cyanobacterium "Arthrospira (Spirulina) maxima". Applied and Environmental Microbiology. 74:6102-6113. AbstractWebsite
Environmental and nutritional conditions that optimize the yield of hydrogen (H-2) from water using a two-step photosynthesis/ fermentation (P/F) process are reported for the hypercarbonate-requiring cyanobacterium "Arthrospira maxima." Our observations lead to four main conclusions broadly applicable to fermentative H-2 production by bacteria: (i) anaerobic H-2 production in the dark from whole cells catalyzed by a bidirectional [NiFe] hydrogenase is demonstrated to occur in two temporal phases involving two distinct metabolic processes that are linked to prior light-dependent production of NADPH (photosynthetic) and dark/anaerobic production of NADH (fermentative), respectively; (ii) H-2 evolution from these reductants represents a major pathway for energy production (ATP) during fermentation by regenerating NAD(+) essential for glycolysis of glycogen and catabolism of other substrates; (iii) nitrate removal during fermentative H-2 evolution is shown to produce an immediate and large stimulation of H-2, as nitrate is a competing substrate for consumption of NAD(P) H, which is distinct from its slower effect of stimulating glycogen accumulation; (iv) environmental and nutritional conditions that increase anaerobic ATP production, prior glycogen accumulation (in the light), and the intracellular reduction potential (NADH/NAD(+) ratio) are shown to be the key variables for elevating H-2 evolution. Optimization of these conditions and culture age increases the H-2 yield from a single P/F cycle using concentrated cells to 36 ml of H-2/g (dry weight) and a maximum 18% H-2 in the headspace. H-2 yield was found to be limited by the hydrogenase-mediated H-2 uptake reaction.
Messing, J, Dooner HK.  2006.  Organization and variability of the maize genome. Current opinion in plant biology. 9:157-63. AbstractWebsite
With a size approximating that of the human genome, the maize genome is about to become the largest plant genome yet sequenced. Contributing to that size are a whole-genome duplication event and a retrotransposition explosion that produced a large amount of repetitive DNA. This DNA is greatly under-represented in cDNA collections, so analysis of the maize transcriptome has been an expedient way of assessing the gene content of maize. Over 2 million maize cDNA sequences are now available, making maize the third most widely studied organism, behind mouse and man. To date, the sequencing of large-sized DNA clones has been largely driven by the genetic interests of different investigators. The recent construction of a physical map that is anchored to the genetic map will aid immensely in the maize genome-sequencing effort. However, studies showing that the repetitive DNA component is highly polymorphic among maize inbred lines point to the need to sample vertically a few specific regions of the genome to evaluate the extent and importance of this variability.
Messing, J, Dooner HK.  2006.  Organization and variability of the maize genome. Curr. Opin. Plant Biol.. 9:157–163. Abstract
With a size approximating that of the human genome, the maize genome is about to become the largest plant genome yet sequenced. Contributing to that size are a whole-genome duplication event and a retrotransposition explosion that produced a large amount of repetitive DNA. This DNA is greatly under-represented in cDNA collections, so analysis of the maize transcriptome has been an expedient way of assessing the gene content of maize. Over 2 million maize cDNA sequences are now available, making maize the third most widely studied organism, behind mouse and man. To date, the sequencing of large-sized DNA clones has been largely driven by the genetic interests of different investigators. The recent construction of a physical map that is anchored to the genetic map will aid immensely in the maize genome-sequencing effort. However, studies showing that the repetitive DNA component is highly polymorphic among maize inbred lines point to the need to sample vertically a few specific regions of the genome to evaluate the extent and importance of this variability.
Xu, JH, Messing J.  2008.  Organization of the prolamin gene family provides insight into the evolution of the maize genome and gene duplications in grass species. Proc Natl Acad Sci U S A. 105:14330-5. AbstractWebsite
Zea mays, commonly known as corn, is perhaps the most greatly produced crop in terms of tonnage and a major food, feed, and biofuel resource. Here we analyzed its prolamin gene family, encoding the major seed storage proteins, as a model for gene evolution by syntenic alignments with sorghum and rice, two genomes that have been sequenced recently. Because a high-density gene map has been constructed for maize inbred B73, all prolamin gene copies can be identified in their chromosomal context. Alignment of respective chromosomal regions of these species via conserved genes allow us to identify the pedigree of prolamin gene copies in space and time. Its youngest and largest gene family, the alpha prolamins, arose about 22-26 million years ago (Mya) after the split of the Panicoideae (including maize, sorghum, and millet) from the Pooideae (including wheat, barley, and oats) and Oryzoideae (rice). The first dispersal of alpha prolamin gene copies occurred before the split of the progenitors of maize and sorghum about 11.9 Mya. One of the two progenitors of maize gained a new alpha zein locus, absent in the other lineage, to form a nonduplicated locus in maize after allotetraplodization about 4.8 Mya. But dispersed copies gave rise to tandem duplications through uneven expansion and gene silencing of this gene family in maize and sorghum, possibly because of maize's greater recombination and mutation rates resulting from its diploidization process. Interestingly, new gene loci in maize represent junctions of ancestral chromosome fragments and sites of new centromeres in sorghum and rice.
Grammont, M, Irvine KD.  2002.  Organizer activity of the polar cells during Drosophila oogenesis. Development. 129:5131-40. AbstractWebsite
Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and are specified by the activation of Notch. We have investigated their role in follicle patterning by creating clones of cells mutant for the Notch modulator fringe. This genetic ablation of polar cells results in cell fate defects within surrounding follicle cells. At the anterior, the border cells, the immediately adjacent follicle cell fate, are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. Our observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis.
Harrison-McMonagle, P, Denissova N, Martínez-Hackert E, Ebright RH, Stock AM.  1999.  Orientation of OmpR monomers within an OmpR:DNA complex determined by DNA affinity cleaving.. Journal of molecular biology. 285(2):555-66. Abstract
Escherichia coli OmpR is a transcription factor that regulates the differential expression of the porin genes ompF and ompC. Phosphorylated OmpR binds as a dimer to a 20-bp region of DNA consisting of two tandemly arranged 10-bp half-sites. Expression of the ompF gene is achieved by the hierarchical occupation of three adjacent 20-bp binding sites, designated F1, F2, and F3 and a distally located site, F4. Despite genetic, biochemical, and structural studies, specific details of the interaction between phosphorylated OmpR and the DNA remain unknown. We have linked the DNA cleaving moiety o-phenanthroline-copper to eight different sites within the DNA binding domain of OmpR in order to determine the orientation of the two OmpR monomers in the OmpR:F1 complex. Five of the resulting conjugates exhibited DNA cleaving activity, and four of these yielded patterns that could be used to construct a model of the OmpR:F1 complex. We propose that OmpR binds asymmetrically to the F1 site as a tandemly arranged dimer with each monomer having its recognition helix in the major groove. The N-terminal end of the recognition helix is promoter-proximal and flanked by "wings" W1 and W2 positioned proximally and distally, respectively, to the transcription start site of ompF. We further propose that the C-terminal end of the recognition helix makes the most extensive contacts with DNA and predict bases within the F1 site that are sufficiently close to be contacted by the recognition helix.
Shin, JA, Ebright RH, Dervan PB.  1991.  Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving.. Nucleic acids research. 19(19):5233-6. Abstract
Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein. Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP [Boelens, R., Scheek, R., van Boom, J. and Kaptein, R., J. Mol. Biol. 193, 1987, 213-216]. In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method. The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR. We have formed the complex between the modified protein and the left DNA half site for LacR. The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.
Dismukes, GC, Blankenship RE.  2005.  The origin and evolution of photosynthetic oxygen production. Photosystem Ii. 22:683-695.Website
Dismukes, GC, Klimov VV, Baranov SV, Kozlov YN, Dasgupta J, Tyryshkin A.  2001.  The origin of atmospheric oxygen on Earth: The innovation of oxygenic photosynthesis. Proceedings of the National Academy of Sciences of the United States of America. 98:2170-2175. AbstractWebsite
The evolution of O-2-producing cyanobacteria that use water as terminal reductant transformed Earth's atmosphere to one suitable for the evolution of aerobic metabolism and complex life. The innovation of water oxidation freed photosynthesis to invade new environments and visibly changed the face of the Earth. We offer a new hypothesis for how this process evolved, which identifies two critical roles for carbon dioxide in the Archean period. First, we present a thermodynamic analysis showing that bicarbonate (formed by dissolution of CO2) is a more efficient alternative substrate than water for O-2 production by oxygenic phototrophs. This analysis clarifies the origin of the long debated "bicarbonate effect" on photosynthetic O-2 production. We propose that bicarbonate was the thermodynamically preferred reductant before water in the evolution of oxygenic photosynthesis. Second, we have examined the speciation of manganese(II) and bicarbonate in water, and find that they form Mn-bicarbonate clusters as the major species under conditions that model the chemistry of the Archean sea. These clusters have been found to be highly efficient precursors for the assembly of the tetramanganese-oxide core of the water-oxidizing enzyme during biogenesis. We show that these clusters can be oxidized at electrochemical potentials that are accessible to anoxygenic phototrophs and thus the most likely building blocks for assembly of the first O-2 evolving photoreaction center, most likely originating from green nonsulfur bacteria before the evolution of cyanobacteria.
Gumienny, T, Padgett R.  2002.  The other side of TGF-β superfamily signal regulation: thinking outside the cell. Trends Endocrinol Metab. 13:295-299. AbstractWebsite
The transforming growth factor beta (TGF-beta) superfamily of paracrine and autocrine signaling molecules regulates a vast array of developmental and homeostatic processes and is itself exquisitely regulated. The misregulation of these molecules often results in cancer and other diseases. Here, we focus on new research that explores how TGF-beta superfamily signaling is controlled between the secreting cell and the target cell. Regulation can occur upon ligand secretion (in a latent protein complex) and in the creation of signaling gradients. Proteins in the extracellular milieu sequester ligand away from or facilitate ligand binding to receptor serine kinases. Ligands even positively regulate their own negative regulators. Studies of how TGF-beta signaling is regulated extracellularly have broadened our understanding of TGF-beta pathways, and could provide clues to our understanding and treatment of diseases resulting from misregulation of these pathways.
Xiang, X, Wu Y, Planta J, Messing J, Leustek T.  2017.  Overexpression of serine acetyltransferase in maize leaves increases seed-specific methionine-rich zeins. Plant biotechnology journal. Abstract
Maize kernels do not contain enough of the essential sulphur-amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)-amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S-assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S-assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell-specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12-fold higher SAT activity without negative impact on growth. S-assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10-kDa δ-zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40-fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high-Met zein accumulation. Moreover, SAT overcomes the shortage of S-amino acids that limits the expression and accumulation of high-Met zeins during kernel development.
Yamamuro, C, Miki D, Zheng Z, Wang J, Dong J, Zhu JK.  2014.  Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation.. Nature Commun.. 5(5):4062.
Carrell, TG, Cohen S, Dismukes CG.  2002.  Oxidative catalysis by Mn4O46+ cubane complexes. Journal of Molecular Catalysis A: Chemical. 187:3-15.Website
Kim, YI, Vinyard DJ, Ananyev GM, Dismukes CG, Golden SS.  2012.  Oxidized quinones signal onset of darkness directly to the cyanobacterial circadian oscillator.. Proceedings of the National Academy of Sciences of the United States of America. 109(44):17765-9. Abstract
Synchronization of the circadian clock in cyanobacteria with the day/night cycle proceeds without an obvious photoreceptor, leaving open the question of its specific mechanism. The circadian oscillator can be reconstituted in vitro, where the activities of two of its proteins, KaiA and KaiC, are affected by metabolites that reflect photosynthetic activity: KaiC phosphorylation is directly influenced by the ATP/ADP ratio, and KaiA stimulation of KaiC phosphorylation is blocked by oxidized, but not reduced, quinones. Manipulation of the ATP/ADP ratio can reset the timing of KaiC phosphorylation peaks in the reconstituted in vitro oscillator. Here, we show that pulses of oxidized quinones reset the cyanobacterial circadian clock both in vitro and in vivo. Onset of darkness causes an abrupt oxidation of the plastoquinone pool in vivo, which is in contrast to a gradual decrease in the ATP/ADP ratio that falls over the course of hours until the onset of light. Thus, these two metabolic measures of photosynthetic activity act in concert to signal both the onset and duration of darkness to the cyanobacterial clock.
Ananyev, GM, Gates C, Dismukes GC.  2016.  The Oxygen quantum yield in diverse algae and cyanobacteria is controlled by partitioning of flux between linear and cyclic electron flow within photosystem II.. Biochim Biophys Acta.. 1857(9):1380-1391. Abstract
We have measured flash-induced oxygen quantum yields (O2-QYs) and primary charge separation (Chl variable fluorescence yield, Fv/Fm) in vivo among phylogenetically diverse microalgae and cyanobacteria. Higher O2-QYs can be attained in cells by releasing constraints on charge transfer at the Photosystem II (PSII) acceptor side by adding membrane-permeable benzoquinone (BQ) derivatives that oxidize plastosemiquinone QB- and QBH2. This method allows uncoupling PSII turnover from its natural regulation in living cells, without artifacts of isolating PSII complexes. This approach reveals different extents of regulation across species, controlled at the QB- acceptor site. Arthrospira maxima is confirmed as the most efficient PSII-WOC (water oxidizing complex) and exhibits the least regulation of flux. Thermosynechococcus elongatus exhibits an O2-QY of 30%, suggesting strong downregulation. WOC cycle simulations with the most accurate model (VZAD) show that a light-driven backward transition (net addition of an electron to the WOC, distinct from recombination) occurs in up to 25% of native PSIIs in the S2 and S3 states, while adding BQ prevents backward transitions and increases the lifetime of S2 and S3 by 10-fold. Backward transitions occur in PSIIs that have plastosemiquinone radicals in the QB site and are postulated to be physiologically regulated pathways for storing light energy as proton gradient through direct PSII-cyclic electron flow (PSII-CEF). PSII-CEF is independent of classical PSI/cyt-b6f-CEF and provides an alternative proton translocation pathway for energy conversion. PSII-CEF enables variable fluxes between linear and cyclic electron pathways, thus accommodating species-dependent needs for redox and ion-gradient energy sources powered by a single photosystem.