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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.
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Berdygulova, Z, Esyunina D, Miropolskaya N, Mukhamedyarov D, Kuznedelov K, Nickels BE, Severinov K, Kulbachinskiy A, Minakhin L.  2012.  A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing.. Nucleic Acids Research. Abstract
Gp39, a small protein encoded by Thermus thermophilus phage P23-45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.
Bilder, D, Irvine KD.  2017.  Taking Stock of the Drosophila Research Ecosystem.. Genetics. 206:1227-1236. Abstract
With a century-old history of fundamental discoveries, the fruit fly has long been a favored experimental organism for a wide range of scientific inquiries. But Drosophila is not a "legacy" model organism; technical and intellectual innovations continue to revitalize fly research and drive advances in our understanding of conserved mechanisms of animal biology. Here, we provide an overview of this "ecosystem" and discuss how to address emerging challenges to ensure its continued productivity. Drosophila researchers are fortunate to have a sophisticated and ever-growing toolkit for the analysis of gene function. Access to these tools depends upon continued support for both physical and informational resources. Uncertainty regarding stable support for bioinformatic databases is a particular concern, at a time when there is the need to make the vast knowledge of functional biology provided by this model animal accessible to scientists studying other organisms. Communication and advocacy efforts will promote appreciation of the value of the fly in delivering biomedically important insights. Well-tended traditions of large-scale tool development, open sharing of reagents, and community engagement provide a strong basis for coordinated and proactive initiatives to improve the fly research ecosystem. Overall, there has never been a better time to be a fly pusher.
Burrows, EH, Bennette NB, Carrieri D, Dixon JL, Brinker A, Frada M, Bakdassabim S N, Falkowski PG, Dismukes GC.  2012.  Dynamics of Lipid Biosynthesis and Redistribution in the Marine Diatom Phaeodactylum tricornutum under Nitrate Deprivation. Bioenerg. Res. 5:876-885. Abstract
One approach to achieve continuous overproduction of lipids in microalgal “cell factories” relies upon depletion or removal of nutrients that act as competing electron sinks (e.g., nitrate and sulfate). However, this strategy can only be effective for bioenergy applications if lipid is synthesized primarily de novo (from CO2 fixation) rather than from the breakdown and interconversion of essential cellular components. In the marine diatom, Phaeodactylum tricornutum, it was determined, using 13C-bicarbonate, that cell growth in nitrate (NO 3 − )-deprived cultures resulted predominantly in de novo lipid synthesis (60 % over 3 days), and this new lipid consisted primarily of triacylglycerides (TAGs). Nearly complete preservation of 12C occurred in all previously existing TAGs in NO 3 − -deprived cultures and thus, further TAG accumulation would not be expected from inhibition of TAG lipolysis. In contrast, both high turnover and depletion of membrane lipids, phosphatidylcholines (PCs), were observed in NO 3 − -deprived cultures (both the headgroups and fatty acid chains), while less turnover was observed in NO 3 − replete cultures. Liquid chromatography-tandem mass spectrometry mass spectra and 13C labeling patterns of PC headgroups provided insight into lipid synthesis in marine diatoms, including suggestion of an internal pool of glycine betaine that feeds choline synthesis. It was also observed that 16C fatty acid chains incorporated into TAGs and PCs contained an average of 14 13C carbons, indicating substantial incorporation of 13C-bicarbonate into fatty acid chains under both nutrient states.
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Deibert, BJ, Zhang J, Smith PF, Chapman KW, Rangan S, Banerjee D, Tan K, Wang H, Pasguale N, Chen F et al..  2015.  Surface and Structural Investigation of a MnOx Birnessite-Type Water Oxidation Catalyst Formed under Photocatalytic Conditions. Chemistry. 21(40):14218-14228. Abstract
Catalytically active MnOx species have been reported to form in situ from various Mn-complexes during electrocatalytic and solution-based water oxidation when employing cerium(IV) ammonium ammonium nitrate (CAN) oxidant as a sacrificial reagent. The full structural characterization of these oxides may be complicated by the presence of support material and lack of a pure bulk phase. For the first time, we show that highly active MnOx catalysts form without supports in situ under photocatalytic conditions. Our most active (4)MnOx catalyst (∼0.84 mmol O2  mol Mn(-1) s(-1)) forms from a Mn4O4 bearing a metal-organic framework. (4)MnOx is characterized by pair distribution function analysis (PDF), Raman spectroscopy, and HR-TEM as a disordered, layered Mn-oxide with high surface area (216 m(2) g(-1)) and small regions of crystallinity and layer flexibility. In contrast, the (S)MnOx formed from Mn(2+) salt gives an amorphous species of lower surface area (80 m(2) g(-1)) and lower activity (∼0.15 mmol O2  mol Mn(-1) s(-1)). We compare these catalysts to crystalline hexagonal birnessite, which activates under the same conditions. Full deconvolution of the XPS Mn2p3/2 core levels detects enriched Mn(3+) and Mn(2+) content on the surfaces, which indicates possible disproportionation/comproportionation surface equilibria.
Dooner, HK, He L.  2014.  Polarized gene conversion at the bz locus of maize.. Proc Natl Acad Sci USA. 111(38):13918-23. Abstract
Nucleotide diversity is greater in maize than in most organisms studied to date, so allelic pairs in a hybrid tend to be highly polymorphic. Most recombination events between such pairs of maize polymorphic alleles are crossovers. However, intragenic recombination events not associated with flanking marker exchange, corresponding to noncrossover gene conversions, predominate between alleles derived from the same progenitor. In these dimorphic heterozygotes, the two alleles differ only at the two mutant sites between which recombination is being measured. To investigate whether gene conversion at the bz locus is polarized, two large diallel crossing matrices involving mutant sites spread across the bz gene were performed and more than 2,500 intragenic recombinants were scored. In both diallels, around 90% of recombinants could be accounted for by gene conversion. Furthermore, conversion exhibited a striking polarity, with sites located within 150 bp of the start and stop codons converting more frequently than sites located in the middle of the gene. The implications of these findings are discussed with reference to recent data from genome-wide studies in other plants.
Dooner, HK, Weil CF.  2013.  Transposons and gene creation. Molecular Genetics and Epigenetics of Plant Transposons. :143-167.
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Gates, C, Ananyev GM, Dismukes C.  2016.  The strontium inorganic mutant of the water oxidizing center (CaMn4O5) of PSII improves WOC efficiency but slows electron flux through the terminal acceptors.. Biochim Biophys Acta.. 1857(9):1550-1560. Abstract
Herein we extend prior studies of biosynthetic strontium replacement of calcium in PSII-WOC core particles to characterize whole cells. Previous studies of Thermosynechococcus elongatus found a lower rate of light-saturated O2 from isolated PSII-WOC(Sr) cores and 5–8 × slower rate of oxygen release. We find similar properties in whole cells, and show it is due to a 20% larger Arrhenius activation barrier for O2 evolution. Cellular adaptation to the sluggish PSII-WOC(Sr) cycle occurs in which flux through the QAQB acceptor gate becomes limiting for turnover rate in vivo. Benzoquinone derivatives that bind to QB site remove this kinetic chokepoint yielding 31% greater O2 quantum yield (QY) of PSII-WOC(Sr) vs. PSII-WOC(Ca). QY and efficiency of the WOC(Sr) catalytic cycle are greatly improved at low light flux, due to fewer misses and backward transitions and 3-fold longer lifetime of the unstable S3 state, attributed to greater thermodynamic stabilization of the WOC(Sr) relative to the photoactive tyrosine YZ. More linear and less cyclic electron flow through PSII occurs per PSII-WOC(Sr). The organismal response to the more active PSII centers in Sr-grown cells at 45 °C is to lower the number of active PSII-WOC per Chl, producing comparable oxygen and energy per cell. We conclude that redox and protonic energy fluxes created by PSII are primary determinants for optimal growth rate of T. elongatus. We further conclude that the (Sr-favored) intermediate-spin S = 5/2 form of the S2 state is the active form in the catalytic cycle relative to the low-spin S = 1/2 form.
Guerra, LT, Xu Y, Bennette N, McNeely K, Bryant DA, Dismukes GC.  2013.  Natural osmolytes are much less effective substrates than glycogen for catabolic energy production in the marine cyanobacterium Synechococcus sp. strain PCC 7002 .. J. Biotechnol.. 166:65-75. Abstract
ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.
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Hawkins, JS, Delgado V, Feng L, Carlise M, Dooner HK, Bennetzen JL.  2014.  Variation in allelic expression associated with a recombination hotspot in Zea mays.. The Plant Journal, DOI: 10.1111/tpj.12537. Abstract
Gene expression is a complex process, requiring precise spatial and temporal regulation of transcription factor activity; however, modifications of individual cis- and trans-acting modules can be molded by natural selection to create a sizeable number of novel phenotypes. Results from decades of research indicate that developmental and phenotypic divergence among eukaryotic organisms is driven primarily by variation in levels of gene expression that are dictated by mutations either in structural or regulatory regions of genes. The relative contributions and interplay of cis- and trans-acting regulatory factors to this evolutionary process, however, remain poorly understood. Analysis of 8 genes in the Bz1-Sh1 interval of maize indicates significant allele-specific expression biases in at least one tissue for all genes, ranging from 1.3-fold to 36-fold. All detected effects were cis-regulatory in nature, although genetic background may also influence the level of expression bias and tissue specificity for some allelic combinations. Most allelic pairs exhibited the same direction and approximate intensity of bias across all four tissues; however, a subset of allelic pairs show alternating dominance across different tissue types or variation in the degree of bias in different tissues. In addition, the genes showing the most striking levels of allelic bias co-localize with a previously described recombination hotspot in this region, suggesting a naturally occurring genetic mechanism for creating regulatory variability for a subset of plant genes that may ultimately lead to evolutionary diversification.
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Ibar, C, Kirichenko E, Keepers B, Enners E, Fleisch K, Irvine KD.  2018.  Tension-dependent regulation of mammalian Hippo signaling through LIMD1.. J Cell Sci. 131:jcs214700. Abstract
Hippo signaling is regulated by biochemical and biomechanical cues that influence the cytoskeleton, but the mechanisms that mediate this have remained unclear. We show that all three mammalian Ajuba family proteins - AJUBA, LIMD1 and WTIP - exhibit tension-dependent localization to adherens junctions, and that both LATS family proteins, LATS1 and LATS2, exhibit an overlapping tension-dependent junctional localization. This localization of Ajuba and LATS family proteins is also influenced by cell density, and by Rho activation. We establish that junctional localization of LATS kinases requires LIMD1, and that LIMD1 is also specifically required for the regulation of LATS kinases and YAP1 by Rho. Our results identify a biomechanical pathway that contributes to regulation of mammalian Hippo signaling, establish that this occurs through tension-dependent LIMD1-mediated recruitment and inhibition of LATS kinases in junctional complexes, and identify roles for this pathway in both Rho-mediated and density-dependent regulation of Hippo signaling.
Irvine, KD, Shraiman BI.  2017.  Mechanical control of growth: ideas, facts and challenges.. Development. 144:4238-4248. Abstract
In his classic book On Growth and Form, D'Arcy Thompson discussed the necessity of a physical and mathematical approach to understanding the relationship between growth and form. The past century has seen extraordinary advances in our understanding of biological components and processes contributing to organismal morphogenesis, but the mathematical and physical principles involved have not received comparable attention. The most obvious entry of physics into morphogenesis is via tissue mechanics. In this Review, we discuss the fundamental role of mechanical interactions between cells induced by growth in shaping a tissue. Non-uniform growth can lead to accumulation of mechanical stress, which in the context of two-dimensional sheets of tissue can specify the shape it assumes in three dimensions. A special class of growth patterns - conformal growth - does not lead to the accumulation of stress and can generate a rich variety of planar tissue shapes. Conversely, mechanical stress can provide a regulatory feedback signal into the growth control circuit. Both theory and experiment support a key role for mechanical interactions in shaping tissues and, via mechanical feedback, controlling epithelial growth.
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Krishnan, A, Kumaraswamy GK, Vinyard DJ, Gu H, Ananyev GM, Posewitz MZ, Dismukes GC.  2015.  Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant.. Plant J. 81(6):947-960. Abstract
Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.
Krishnan, A, Zhang S, Liu Y, Tadmori KA, Bryant DA, Dismukes GC.  2016.  Consequences of ccmR deletion on respiration, fermentation and H2 metabolism in cyanobacterium Synechococcus sp. PCC 7002. Biotechnol Bioeng. Abstract
CcmR, a LysR-type transcriptional regulator, represses the genes encoding components of the high-affinity carbon concentration mechanism in cyanobacteria. Unexpectedly, deletion of the ccmR gene was found to alter the expression of the terminal oxidase and fermentative genes, especially the hydrogenase operon in the cyanobacterium Synechococcus sp. PCC 7002. Consistent with the transcriptomic data, the deletion strain exhibits flux increases (30-50%) in both aerobic O2 respiration and anaerobic H2 evolution. To understand how CcmR influences anaerobic metabolism, the kinetics of autofermentation were investigated following photoautotrophic growth. The autofermentative H2 yield increased by 50% in the CcmR deletion strain compared to the wild-type strain, and increased to 160% (within 20 h) upon continuous removal of H2 from the medium ("milking") to suppress uptake. Consistent with this greater reductant flux to H2 , the mutant excreted less lactate during autofermentation (NAD(P)H consuming pathway). To enhance the rate of NADH production during anaerobic metabolism, the ccmR mutant was engineered to introduce GAPDH overexpression (more NADH production) and LDH deletion (less NADH consumption). The triple mutant (ccmR deletion + GAPDH overexpression + LDH deletion) showed 6-8-fold greater H2 yield than the WT strain, achieving conversion rates of 17 nmol 108 cells-1 h-1 and yield of 0.87 H2 per glucose equivalent (8.9% theoretical maximum). Simultaneous monitoring of the intracellular NAD(P)H concentration and H2 production rate by these mutants reveals an inverse correspondence between these variables indicating hydrogenase-dependent H2 production as a major sink for consuming NAD(P)H in preference to excretion of reduced carbon as lactate during fermentation.
Kumaraswamy, GK, Guerra T, Qian X, Zhang S, Bryant DA, Dismukes GC.  2013.  Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: metabolic engineering of NAD+-dependent GAPDH. Energy Environ. Sci.. 6:3722-3731. AbstractWebsite
Catabolism of glycogen stored by cyanobacteria occurs during anaerobic auto-fermentation and produces a range of C1–C3 fermentation products and hydrogen via hydrogenase. We investigated both augmenting and rerouting this carbon catabolism by engineering the glycolysis pathway at the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH-1), its major regulation site at the nexus of two pathways (Oxidative Pentose Phosphate pathway, OPP, and glycolysis/gluconeogenesis). Null (gap1::aphII) and overexpression (gap1+) strains of Synechococcus sp. strain PCC 7002 were constructed in order to produce more NADPH (via rerouting carbon through OPP) and more NADH (via opening the glycolytic bottleneck), respectively. For gap1::aphII quantitative analyses after four days of dark auto-fermentation showed undiminished glycogen catabolism rate, significant increases of intracellular metabolites in both OPP and upper-glycolysis, decrease in lower-glycolysis intermediates, 5.7-fold increase in NADPH, 2.3-fold increase in hydrogen and 1.25-fold increase in CO2vs. wild type (WT). These changes demonstrate the expected outcome of redirection of carbon catabolism through the OPP pathway with significant stimulation of OPP product yields. The gap1+ strain exhibits a large 17% increase in accumulation of glycogen during the prior photoautotrophic growth stage (gluconeogenesis), in parallel with a 2-fold increase in the total [NAD+ + NADH] pool, foreshadowing an increased catabolic capacity. Indeed, the rate of glycogen catabolism during subsequent dark auto-fermentation increased significantly (58%) vs. WT, resulting in increases in both NADH (4.0-fold) and NADPH (2.9-fold) pools, and terminal fermentation products, hydrogen (3.0-fold) D-lactate (2.3-fold) and acetate (1.4-fold). The overall energy conversion yield over four days from catabolized glycogen to hydrogen increased from 0.6 mole of hydrogen per mole of glucose (WT) to 1.4 (gap1::aphII) and 1.1 (gap1+) under headspace accumulation conditions (without hydrogen milking). These findings demonstrate the significant potential of metabolic engineering for redirecting carbon pathways for carbohydrate catabolism and hydrogen production in cyanobacteria.
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Li, Y., Segal, G., Wang, Q., Dooner HK.  2013.  Gene tagging with engineered Ds elements in maize. Methods in Molecular Biology: Plant Transposable Elements. :83-99.
Li, Y., Dooner HK.  2012.  Helitron Proliferation and Gene-Fragment Capture. Topics in Current Genetics, 24: Plant Transposable Elements- Impact on Genome Structure and Function. :193-227.
Li, Y, Harris L, Dooner HK.  2013.  TED, an autonomous and rare maize transposon of the mutator superfamily with a high gametophytic excision frequency.. The Plant cell. 25(9):3251-65. Abstract
Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor.
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Mao, Y, Sun S, Irvine KD.  2017.  Role and regulation of Yap in KrasG12D-induced lung cancer.. Oncotarget. 8:110877-110889. Abstract
The Hippo pathway and its downstream transcriptional co-activator Yap influence lung cancer, but the nature of the Yap contribution has been unclear. Using a genetically engineered mouse lung cancer model, we show that Yap deletion completely blocks KrasG12D and p53 loss-driven adenocarcinoma initiation and progression, whereas heterozygosity for Yap partially suppresses lung cancer growth and progression. We also characterize Yap expression during tumor progression and find that nuclear Yap can be detected from the earliest stages of lung carcinogenesis, but at levels comparable to that in aveolar type II cells, which are a cell of origin for lung adenocarcinoma. At later stages of tumorigenesis, variations in Yap levels are detected, which correlate with differences in cell proliferation within tumors. Our observations imply that Yap is not directly activated by oncogenic Kras during lung tumorigenesis, but is nonetheless absolutely required for this tumorigenesis, and support Yap as a therapeutic target in lung adenocarcinoma.
Miclaus, M, Wu Y, Xu J, Dooner HK, Messing J.  2011.  The maize high-lysine mutant opaque7 is defective in an acyl-CoA synthetase-like protein.. Genetics. 189:1271-1280.
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Nickels, BE.  2012.  A new way to start: nanoRNA-mediated priming of transcription initiation.. Transcription. 3(6):300-304. Abstract
A recent study provides evidence that RNA polymerase uses 2- to ~4-nt RNAs, species termed "nanoRNAs," to prime transcription initiation in Escherichia coli. Priming of transcription initiation with nanoRNAs represents a previously undocumented component of transcription start site selection and gene expression.
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Oh, H, Slattery M, Ma L, Crofts A, White KP, Mann RS, Irvine KD.  2013.  Genome-wide association of Yorkie with chromatin and chromatin-remodeling complexes. Cell reports. 3:309-18. AbstractWebsite
The Hippo pathway regulates growth through the transcriptional coactivator Yorkie, but how Yorkie promotes transcription remains poorly understood. We address this by characterizing Yorkie's association with chromatin and by identifying nuclear partners that effect transcriptional activation. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF), the Brahma complex, and the Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkie's transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin-binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of messenger RNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. Our studies suggest a widespread role for Yorkie as a regulator of transcription and identify recruitment of the chromatin-modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie.
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Qian, X, Kumaraswamy GK, Zhang S, Gates C, Ananyev GM, Bryant DA, Dismukes GC.  2015.  Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.. Biotechnol Bioeng. 113(5):979-988. Abstract
To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations-both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))-were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.
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Smith, PF, Hunt L, Laursen AB, Sagar V, Kaushik S, Calvinho KU, Marotta G, Mosconi E, De Angelis F, Dismukes GC.  2015.  Water Oxidation by the [Co4O4(OAc)4(py)4](+) Cubium is Initiated by OH(-) Addition.. J Am Chem Soc. 137(49):15460-15468. Abstract
The cobalt cubium Co4O4(OAc)4(py)4(ClO4) (1A(+)) containing the mixed valence [Co4O4](5+) core is shown by multiple spectroscopic methods to react with hydroxide (OH(-)) but not with water molecules to produce O2. The yield of reaction products is stoichiometric (>99.5%): 41A(+) + 4OH(-) → O2 + 2H2O + 41A. By contrast, the structurally homologous cubium Co4O4(trans-OAc)2(bpy)4(ClO4)3, 1B(ClO4)3, produces no O2. EPR/NMR spectroscopies show clean conversion to cubane 1A during O2 evolution with no Co(2+) or Co3O4 side products. Mass spectrometry of the reaction between isotopically labeled μ-(16)O(bridging-oxo) 1A(+) and (18)O-bicarbonate/water shows (1) no exchange of (18)O into the bridging oxos of 1A(+), and (2) (36)O2 is the major product, thus requiring two OH(-) in the reactive intermediate. DFT calculations of solvated intermediates suggest that addition of two OH(-) to 1A(+) via OH(-) insertion into Co-OAc bonds is energetically favored, followed by outer-sphere oxidation to intermediate [1A(OH)2](0). The absence of O2 production by cubium 1B(3+) indicates the reactive intermediate derived from 1A(+) requires gem-1,1-dihydoxo stereochemistry to perform O-O bond formation. Outer-sphere oxidation of this intermediate by 2 equiv of 1A(+) accounts for the final stoichiometry. Collectively, these results and recent literature (Faraday Discuss., doi:10.1039/C5FD00076A and J. Am. Chem. Soc. 2015, 137, 12865-12872) validate the [Co4O4](4+/5+) cubane core as an intrinsic catalyst for oxidation of hydroxide by an inner-sphere mechanism.