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Garner, AL, Rammohan J, Huynh JP, Onder LM, Chen J, Bae B, Jensen D, Weiss LA, Manzano AR, Darst SA et al..  2017.  Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence. Journal of Bacteriology. 199:e00698-16..
Dorsett, D, Eissenberg JC, Misulovin Z, Martens A, Redding B, McKim K.  2005.  Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila. Development. 132:4743-53.Website
McKim, KS, Howell AM, Rose AM.  1988.  The effects of translocations on recombination frequency in Caenorhabditis elegans. Genetics. 120:987-1001.
Kane, NS, Vora M, Varre KJ, Padgett RW.  2016.  Efficient Screening of CRISPR/Cas9-Induced Events in Drosophila using a co-CRISPR Strategy. G3. 7(1):87-93.
Liu, G, Rogers J, Murphy CT, Rongo C.  2011.  EGF signalling activates the ubiquitin proteasome system to modulate C. elegans lifespan. EMBO J. 30:2990-3003. AbstractWebsite
Epidermal growth factor (EGF) signalling regulates growth and differentiation. Here, we examine the function of EGF signalling in Caenorhabditis elegans lifespan. We find that EGF signalling regulates lifespan via the Ras-MAPK pathway and the PLZF transcription factors EOR-1 and EOR-2. As animals enter adulthood, EGF signalling upregulates the expression of genes involved in the ubiquitin proteasome system (UPS), including the Skp1-like protein SKR-5, while downregulating the expression of HSP16-type chaperones. Using reporters for global UPS activity, protein aggregation, and oxidative stress, we find that EGF signalling alters protein homoeostasis in adults by increasing UPS activity and polyubiquitination, while decreasing protein aggregation. We show that SKR-5 and the E3/E4 ligases that comprise the ubiquitin fusion degradation (UFD) complex are required for the increase in UPS activity observed in adults, and that animals that lack SKR-5 or the UFD have reduced lifespans and indications of oxidative stress. We propose that as animals enter fertile adulthood, EGF signalling switches the mechanism for maintaining protein homoeostasis from a chaperone-based approach to an approach involving protein elimination via augmented UPS activity.
Parry, JM, Singson A.  2011.  EGG molecules couple the oocyte-to-embryo transition with cell cycle progression. Results Probl Cell Differ. 53:135–151. Abstract
The oocyte-to-embryo transition is a precisely coordinated process in which an oocyte becomes fertilized and transitions to an embryonic program of events. The molecules involved in this process have not been well studied. Recently, a group of interacting molecules in C. elegans have been described as coordinating the oocyte-to-embryo transition with the advancement of the cell cycle. Genes egg-3, egg-4, and egg-5 represent a small class of regulatory molecules known as protein-tyrosine phosphase-like proteins, which can bind phosphorylated substrates and act as scaffolding molecules or inhibitors. These genes are responsible for coupling the movements and activities of regulatory kinase mbk-2 with advancement of the cell cycle during the oocyte-to-embryo transition.
Shi, A, Chen CC, Banerjee R, Glodowski D, Audhya A, Rongo C, Grant BD.  2010.  EHBP-1 functions with RAB-10 during endocytic recycling in Caenorhabditis elegans. Mol Biol Cell. 21:2930-43. AbstractWebsite
Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans. In a yeast two-hybrid screen for binding partners of RAB-10 we identified EHBP-1, a calponin homology domain (CH) protein, whose mammalian homolog Ehbp1 was previously shown to function during endocytic transport of GLUT4 in adipocytes. In vivo we find that EHBP-1-GFP colocalizes with RFP-RAB-10 on endosomal structures of the intestine and interneurons and that ehbp-1 loss-of-function mutants share with rab-10 mutants specific endosome morphology and cargo localization defects. We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8. These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.
Brimblecombe, R, Bond AM, Dismukes CG, Swiegers GF, Spiccia L.  2009.  Electrochemical investigation of Mn4O4-cubane water-oxidizing clusters. Physical Chemistry Chemical Physics. 11:6441-6449. AbstractWebsite
High valence states in manganese clusters are a key feature of the function of one of the most important catalysts found in nature, the water-oxidizing complex of photosystem II. We describe a detailed electrochemical investigation of two bio-inspired manganese-oxo complexes, [Mn4O4L6] (L = diphenylphosphinate (1) and bis(p-methoxyphenyl)phosphinate (2)), in solution, attached to an electrode surface and suspended within a Nafion film. These complexes contain a cubic [Mn4O4]6+ core stabilized by phosphinate ligands. They have previously been shown to be active and durable photocatalysts for the oxidation of water to dioxygen. A comparison of catalytic photocurrent generated by films deposited by two methods of electrode immobilization reveals that doping of the catalyst in Nafion results in higher photocurrent than was observed for a solid layer of cubane on an electrode surface. In dichloromethane solution, and under conditions of cyclic voltammetry, the one-electron oxidation processes 1/1+ and 2/2+ were found to be reversible and quasi-reversible, respectively. Some decomposition of 1+ and 2+ was detected on the longer timescale of bulk electrolysis. Both compounds also undergo a two-electron, chemically irreversible reduction in dichloromethane, with a mechanism that is dependent on scan rate and influenced by the presence of a proton donor. When immersed in aqueous electrolyte, the reduction process exhibits a limited level of chemical reversibility. These data provide insights into the catalytic operation of these molecules during photo-assisted electrolysis of water and highlight the importance of the strongly electron-donating ligand environment about the manganese ions in the ability of the cubanes to photocatalyze water oxidation at low overpotentials.
Lund, G, Messing J, Viotti A.  1995.  Endosperm-specific demethylation and activation of specific alleles of alpha-tubulin genes of Zea mays L. Molecular & general genetics : MGG. 246:716-22. AbstractWebsite
We have investigated the methylation status of the alpha-tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the alpha-tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub alpha 2 and tub alpha 4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two alpha-tubulin genes, tub alpha 3 and tub alpha 4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.
Liao, JC, Messing J.  2012.  Energy biotechnology. Current opinion in biotechnology. 23(3):287-9.Website
Hart, B, Mathias JR, Ott D, McNaughton L, Anderson JS, Vershon AK, Baxter SM.  2002.  Engineered Improvements in DNA-binding Function of the MATa1 Homeodomain Reveal Structural Changes Involved in Combinatorial Control. J Mol Biol. 316:247-256. Abstract
We have engineered enhanced DNA-binding function into the a1 homeodomain by making changes in a loop distant from the DNA-binding surface. Comparison of the free and bound a1 structures suggested a mechanism linking van der Waals stacking changes in this loop to the ordering of a final turn in the DNA-binding helix of a1. Inspection of the protein sequence revealed striking differences in amino acid identity at positions 24 and 25 compared to related homeodomain proteins. These positions lie in the loop connecting helix-1 and helix-2, which is involved in heterodimerization with the alpha 2 protein. A series of single and double amino acid substitutions (a1-Q24R, a1-S25Y, a1-S25F and a1-Q24R/S25Y) were engineered, expressed and purified for biochemical and biophysical study. Calorimetric measurements and HSQC NMR spectra confirm that the engineered variants are folded and are equally or more stable than the wild-type a1 homeodomain. NMR analysis of a1-Q24R/S25Y demonstrates that the DNA recognition helix (helix-3) is extended by at least one turn as a result of the changes in the loop connecting helix-1 and helix-2. As shown by EMSA, the engineered variants bind DNA with enhanced affinity (16-fold) in the absence of the alpha 2 cofactor and the variant alpha 2/a1 heterodimers bind cognate DNA with specificity and affinity reflective of the enhanced a1 binding affinity. Importantly, in vivo assays demonstrate that the a1-Q24R/S25Y protein binds with fivefold greater affinity than wild-type a1 and is able to partially suppress defects in repression by alpha 2 mutants. As a result of these studies, we show how subtle differences in residues at a surface distant from the functional site code for a conformational switch that allows the a1 homeodomain to become active in DNA binding in association with its cofactor alpha 2.
Vinyard, DJ, Gimpel J, Ananyev GM, Mayfield SP, Dismukes CG.  2014.  Engineered Photosystem II reaction centers optimize photochemistry versus photoprotection at different solar intensities.. Journal of the American Chemical Society. 136(10):4048-55. Abstract
The D1 protein of Photosystem II (PSII) provides most of the ligating amino acid residues for the Mn4CaO5 water-oxidizing complex (WOC) and half of the reaction center cofactors, and it is present as two isoforms in the cyanobacterium Synechococcus elongatus PCC 7942. These isoforms, D1:1 and D1:2, confer functional advantages for photosynthetic growth at low and high light intensities, respectively. D1:1, D1:2, and seven point mutations in the D1:2 background that are native to D1:1 were expressed in the green alga Chlamydomonas reinhardtii. We used these nine strains to show that those strains that confer a higher yield of PSII charge separation under light-limiting conditions (where charge recombination is significant) have less efficient photochemical turnover, measured in terms of both a lower WOC turnover probability and a longer WOC cycle period. Conversely, these same strains under light saturation (where charge recombination does not compete) confer a correspondingly faster O2 evolution rate and greater protection against photoinhibition. Taken together, the data clearly establish that PSII primary charge separation is a trade-off between photochemical productivity (water oxidation and plastoquinone reduction) and charge recombination (photoprotection). These trade-offs add up to a significant growth advantage for the two natural isoforms. These insights provide fundamental design principles for engineering of PSII reaction centers with optimal photochemical efficiencies for growth at low versus high light intensities.
Planta, J, Xiang X, Leustek T, Messing J.  2017.  Engineering sulfur storage in maize seed proteins without apparent yield loss.. Proceedings of the National Academy of Sciences of the United States of America. 114(43):11386-11391. Abstract
Sulfur assimilation may limit the pool of methionine and cysteine available for incorporation into zeins, the major seed storage proteins in maize. This hypothesis was tested by producing transgenic maize with deregulated sulfate reduction capacity achieved through leaf-specific expression of the Escherichia coli enzyme 3'-phosphoadenosine-5'-phosphosulfate reductase (EcPAPR) that resulted in higher methionine accumulation in seeds. The transgenic kernels have higher expression of the methionine-rich 10-kDa δ-zein and total protein sulfur without reduction of other zeins. This overall increase in the expression of the S-rich zeins describes a facet of regulation of these proteins under enhanced sulfur assimilation. Transgenic line PE5 accumulates 57.6% more kernel methionine than the high-methionine inbred line B101. In feeding trials with chicks, PE5 maize promotes significant weight gain compared with nontransgenic kernels. Therefore, increased source strength can improve the nutritional value of maize without apparent yield loss and may significantly reduce the cost of feed supplementation.
Maliga, P, Svab Z.  2011.  Engineering the plastid genome of Nicotiana sylvestris, a diploid model species for plastid genetics. Methods in Molecular Biology. 701:37-50. AbstractWebsite
The plastids of higher plants have their own approximately 120-160-kb genome that is present in 1,000-10,000 copies per cell. Engineering of the plastid genome (ptDNA) is based on homologous recombination between the plastid genome and cloned ptDNA sequences in the vector. A uniform population of engineered ptDNA is obtained by selection for marker genes encoded in the vectors. Manipulations of ptDNA include (1) insertion of transgenes in intergenic regions; (2) posttransformation excision of marker genes to obtain marker-free plants; (3) gene knockouts and gene knockdowns, and (4) cotransformation with multiple plasmids to introduce nonselected genes without physical linkage to marker genes. Most experiments on plastome engineering have been carried out in the allotetraploid Nicotiana tabacum. We report here for the first time plastid transformation in Nicotiana sylvestris, a diploid ornamental species. We demonstrate that the protocols and vectors developed for plastid transformation in N. tabacum are directly applicable to N. sylvestris with the advantage that the N. sylvestris transplastomic lines are suitable for mutant screens.
Ananyev, GM, Skizim NJ, Dismukes CG.  2012.  Enhancing biological hydrogen production from cyanobacteria by removal of excreted products.. Journal of biotechnology. 162(1):97-104. Abstract
Hydrogen is produced by a [NiFe]-hydrogenase in the cyanobacterium Arthrospira (Spirulina) maxima during autofermentation of photosynthetically accumulated glycogen under dark anaerobic conditions. Herein we show that elimination of H₂ backpressure by continuous H₂ removal ("milking") can significantly increase the yield of H₂ in this strain. We show that "milking" by continuous selective consumption of H₂ using an electrochemical cell produces the maximum increase in H₂ yield (11-fold) and H₂ rate (3.4-fold), which is considerably larger than through "milking" by non-selective dilution of the biomass in media (increases H₂ yield 3.7-fold and rate 3.1-fold). Exhaustive autofermentation under electrochemical milking conditions consumes >98% of glycogen and 27.6% of biomass over 7-8 days and extracts 39% of the energy content in glycogen as H₂. Non-selective dilution stimulates H₂ production by shifting intracellular equilibria competing for NADH from excreted products and terminal electron sinks into H₂ production. Adding a mixture of the carbon fermentative products shifts the equilibria towards reactants, resulting in increased intracellular NADH and an increased H₂ yield (1.4-fold). H₂ production is sustained for a period of time up to 7days, after which the PSII activity of the cells decreases by 80-90%, but can be restored by regeneration under photoautotrophic growth.
Carrell TG, Smith PF, Dennes J, Dismukes CG.  2014.  Entropy and enthalpy contributions to the kinetics of proton coupled electron transfer to the Mn4O4(O2PPh2)6 cubane.. Physical chemistry chemical physics : PCCP. 16(24):11843-7. Abstract
The dependence of rate, entropy of activation, and ((1)H/(2)H) kinetic isotope effect for H-atom transfer from a series of p-substituted phenols to cubane Mn4O4L6 (L = O2PPh2) () reveals the activation energy to form the transition state is proportional to the phenolic O-H bond dissociation energy. New implications for water oxidation and charge recombination in photosystem II are described.
Goettel, W, Messing J.  2013.  Epiallele biogenesis in maize. Gene. 516:8-23. AbstractWebsite
We have correlated cytosine methylation of two epialleles, P1-rr and P1-pr, with variation in gene expression and therefore phenotype. The p1 gene in maize encodes a transcription factor that controls phlobaphene pigment accumulation in floral tissues. While cytosine methylation was assayed in various regions spanning 17 kb, the only difference in DNA methylation pattern between the expressed P1-rr allele and the silenced P1-pr allele was detected in a region that consists of a complex arrangement of transposons and adjacent repeats. This region, which comprises the distal enhancer element of P1-rr, is hypermethylated in P1-pr compared to P1-rr. Based on other precedents, we hypothesize that DNA methylation spreads from the transposable elements into the flanking P1-rr enhancer, thereby transcriptionally silencing the gene. Interestingly, P1-pr is reactivated in mutants of the dominant epigenetic modifier Ufo1. DNA methylation in the distal enhancer sequence is significantly reduced, which inversely correlates with increased transcript levels and pigmentation in P1-pr Ufo1 plants. If in general DNA methylation spreads from transposons into adjacent sequences containing regulatory elements for neighboring genes, the corresponding genes could be silenced by chance. Given the large amount of transposable elements in the maize genome, epialleles may be far more frequent than previously estimated.
Rongo, C.  2011.  Epidermal growth factor and aging: A signaling molecule reveals a new eye opening function. Aging. 3(9):1-10. AbstractWebsite
Epidermal Growth Factor (EGF) is known for its role in promoting cell division and cellular differentiation in developing animals, but we know surprising little about what EGF does in vivo in mature adult animals. Here I review EGF signaling, emphasizing several recent studies that uncovered an unexpected role for EGF in promoting longevity and healthspan in mature adult C. elegans. EGF, acting through phospholipase Cγ and the IP3 receptor signaling, maintains pharyngeal and body wall muscle function in aging adults, and delays the accumulation of lipofuscin-enriched aging pigments within intestinal cells. EGF also acts through the Ras/ERK pathway to regulate protein homeostasis by promoting the expression of antioxidant genes, stimulating the activity of the Ubiquitin Proteasome System (UPS), and repressing the expression of small heat shock protein chaperones. The effects of EGF signaling on lifespan are largely independent of Insulin/IGF-like Signaling (IIS), as the effects of EGF signaling mutants on lifespan and heathspan are not affected by mutations in the DAF-2 insulin receptor or the DAF-16 FOXO transcription factor. Nevertheless, these two signal pathways have multiple points of overlap, coordination, and cross regulation. I propose that the IIS and EGF signaling pathways respond to environment and to developmental timing, respectively, so as to coordinate the appropriate physiological strategy that cells use to maintain protein homeostasis.
Ebright, RH, Busby S.  1995.  The Escherichia coli RNA polymerase alpha subunit: structure and function.. Current opinion in genetics & development. 5(2):197-203. Abstract
Recent work has established that the Escherichia coli RNA polymerase alpha subunit consists of an amino-terminal domain containing determinants for interaction with the remainder of RNA polymerase, a carboxy-terminal domain containing determinants for interaction with DNA and interaction with transcriptional activator proteins, and a 13-36 amino acid unstructured and/or flexible linker. These findings suggest a simple, integrated model for the mechanism of involvement of alpha in promoter recognition and transcriptional activation.
Tang, H, Kim Y, Severinov K, Goldfarb A, Ebright RH.  1996.  Escherichia coli RNA polymerase holoenzyme: rapid reconstitution from recombinant alpha, beta, beta', and sigma subunits.. Methods in enzymology. 273:130-4.
Ebright, RH.  1986.  Evidence for a contact between glutamine-18 of lac repressor and base pair 7 of lac operator.. Proceedings of the National Academy of Sciences of the United States of America. 83(2):303-7. Abstract
Glutamine-18 of the lac repressor (lacR) has been substituted by glycine, by serine, and by leucine. The specificities of wild-type lacR and of the three substituted lacR variants have been analyzed with respect to base pairs 5, 6, 7, 8, 9, and 10 of the lac operator (lacO). The data indicate that [Gly18]lacR, [Ser18]lacR, and [Leu18]lacR lose the ability to distinguish between the O+ base pair G . C and the Oc base pairs T . A and A . T at position 7 of lacO (KdOc/KdO+ approximately equal to 1). In contrast, the three substituted variants retain the ability to discriminate O+ from Oc at each other position, by factors of 9 to 37. Therefore, I propose that glutamine-18 contacts base pair 7 of lacO. These data suggest that the interaction between the helix-turn-helix motif and DNA may be very similar or identical in lacR and the catabolite gene activator protein.
Garcia, N, Zhang W, Wu Y, Messing J.  2015.  Evolution of gene expression after gene amplification.. Genome biology and evolution. 7(5):1303-12. AbstractWebsite
We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat-maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that γ-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a γ-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators.
Wang, W, Kerstetter R, Michael TP..  2011.  Evolution of Genome Size in Duckweeds (Lemnaceae).. Journal of Botany.