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Brimblecombe, R, Koo A, Dismukes CG, Swiegers GF, Spiccia L.  2010.  A Tandem Water-Splitting Device Based on a Bio-inspired Manganese Catalyst. ChemSusChem. 3:1146-1150.Website
Dismukes, CG, Brimblecombe R, Koo A, Swiegers GF, Spiccia L.  2010.  A tandem water-splitting device based on a bio-inspired manganese catalyst.. ChemSusChem. 3(10):1146-50.
Radford, SJ, McKim KS.  2016.  Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes.. Journal of visualized experiments : JoVE. (116) Abstract
Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.
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.
Zhang, W, Xu J H, Bennetzen JL, Messing J.  2016.  Teff, an Orphan Cereal in the Chloridoideae, Provides Insights into the Evolution of Storage Proteins in Grasses.. Genome biology and evolution. 8(6):1712-21. Abstract
Seed storage proteins (SSP) in cereals provide essential nutrition for humans and animals. Genes encoding these proteins have undergone rapid evolution in different grass species. To better understand the degree of divergence, we analyzed this gene family in the subfamily Chloridoideae, where the genome of teff (Eragrostis tef) has been sequenced. We find gene duplications, deletions, and rapid mutations in protein-coding sequences. The main SSPs in teff, like other grasses, are prolamins, here called eragrostins. Teff has γ- and δ-prolamins, but has no β-prolamins. One δ-type prolamin (δ1) in teff has higher methionine (33%) levels than in maize (23-25%). The other δ-type prolamin (δ2) has reduced methionine residues (<10%) and is phylogenetically closer to α prolamins. Prolamin δ2 in teff represents an intermediate between δ and α types that appears to have been lost in maize and other Panicoideae, and was replaced by the expansion of α-prolamins. Teff also has considerably larger numbers of α-prolamin genes, which we further divide into five sub-groups, where α2 and α5 represent the most abundant α-prolamins both in number and in expression. In addition, indolines that determine kernel softness are present in teff and the panicoid cereal called foxtail millet (Setaria italica) but not in sorghum or maize, indicating that these genes were only recently lost in some members of the Panicoideae Moreover, this study provides not only information on the evolution of SSPs in the grass family but also the importance of α-globulins in protein aggregation and germplasm divergence.
Pavlova, O, Lavysh D, Klimuk E, Djordjevic M, Ravcheev DA, Gelfand MS, Severinov K, Akulenko N.  2012.  Temporal regulation of gene expression of the Escherichia coli bacteriophage phiEco32. J. Mol. Biol. 416:389-399.
Berdygulova, Z, Westblade LF, Florens L, Chait BT, Ramanculov E, Washburn MP, Darst SA, Severinov K, Minakhin L.  2011.  Temporal regulation of gene expression of the Thermus thermophilus bacteriophage P23-45. J. Mol. Biol.. 405:125-142.
Twombly, V, Blackman RK, Jin H, Graff JM, Padgett RW, Gelbart WM.  1996.  The TGF-beta signaling pathway is essential for Drosophila oogenesis. Development (Cambridge, England). 122:1555-65. AbstractWebsite
We examine roles of signaling by secreted ligands of the TGF-beta family during Drosophila oogenesis. One family member, the DPP ligand encoded by the decapentaplegic (dpp) gene, is required for patterning of anterior eggshell structures. This requirement presumably reflects the expression pattern of dpp in an anterior subset of somatic follicle cells: the centripetally migrating and the nurse cell-associated follicle cells. Similar requirements are also revealed by mutations in the saxophone (sax)-encoded receptor, consistent with the idea that DPP signaling is, at least in part, mediated by the SAX receptor. A loss of germline sax function results in a block in oogenesis associated with egg chamber degeneration and a failure of the transfer of nurse cell contents to the oocyte, indicating that TGF-beta signaling is required for these events. Some phenotypes of sax mutations during oogenesis suggest that SAX responds to at least one other TGF-beta ligand as well in the posterior follicle cells.
Savage-Dunn, C, Padgett RW.  2017.  The TGF-β Family in Caenorhabditis elegans. The Biology of the TGF-β Family.
Padgett, RW, Das P, Krishna S.  1998.  TGF-β signaling, Smads, and tumor suppressors. BioEssays : news and reviews in molecular, cellular and developmental biology. 20:382-90. AbstractWebsite
The transforming growth factor-beta (TGF-beta) superfamily is used throughout animal development for regulating the growth and patterning of many tissue types. During the past few years, rapid progress has been made in deciphering how TGF-beta signals are transduced from outside the cell to the nucleus. This progress is based on biochemical studies in vertebrate systems and a combination of genetic studies in Drosophila and Caenorhabditis elegans. These studies have identified a novel family of signaling proteins, the Smad family. Smads can act positively and be phosphorylated by TGF-beta-like receptors or can act negatively and prevent activation of the positively acting group. The positively acting Smads translocate to the nucleus, bind DNA, and act as transcriptional activators. Thus, genetic and biochemical studies suggest a very simple signaling pathway, in which Smads are the primary downstream participant.
Padgett, RW.  1999.  TGFβ signaling pathways and human diseases. Cancer metastasis reviews. 18:247-59. AbstractWebsite
Recent progress in deciphering the TGFbeta pathway has uncovered a new signaling molecule, the Smads, and with this finding now gives us insights into how TGFbeta-like signals are transmitted from outside the cell to the nucleus. As we learn more about how TGFbeta regulates normal development, we also are gaining insights into diseases that are caused by mis-regulation or mutation of various components of the signaling pathways.
Padgett, RW, Das P, Krishna S.  1998.  TGFβ Signaling, Smads, and Tumor Suppressors. BioEssays. 20:382-390.
Padgett, RW, Reiss M.  2007.  TGFβ superfamily signaling: notes from the desert. Development (Cambridge, England). 134:3565-9. AbstractWebsite
The TGFbeta pathways play crucial roles in many developmental events, as well as contributing to many disease states. To provide a venue for both signaling and developmental research on TGFbeta, a FASEB-sponsored bi-annual meeting was initiated six years ago, the fourth of which was organized by Caroline Hill and Michael O'Connor and took place this July in Tucson, Arizona. The meeting highlighted major advances in our understanding of the structural and biochemical aspects of TGFbeta superfamily signaling, its intersection with other pathways, and its contribution to disease.
Patterson, G, Padgett R.  2000.  TGFβ-related pathways. Roles in Caenorhabditis elegans development. Trends Genet. 16:27-33. AbstractWebsite
Genetic and molecular analysis in Caenorhabditis elegans has produced new insights into how TGF beta-related pathways transduce signals and the developmental processes in which they function. These pathways are essential regulators of dauer formation, body-size determination, male copulatory structures and axonal guidance. Here, we review the insights that have come from standard molecular genetic experiments and discuss how the recently completed genome sequence has contributed to our understanding of these pathways.
Hawley, RS, Irick HA, Zitron AE, Haddox DA, Lohe AR, New C, Whitley MD, Arbel T, Jang JK, McKim KS et al..  1993.  There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology. Developmental Genetics. 13:440-467.
Tadigotla, VR, O Maoiléidigh D, Sengupta AM, Epshtein V, Ebright RH, Nudler E, Ruckenstein AE.  2006.  Thermodynamic and kinetic modeling of transcriptional pausing.. Proceedings of the National Academy of Sciences of the United States of America. 103(12):4439-44. Abstract
We present a statistical mechanics approach for the prediction of backtracked pauses in bacterial transcription elongation derived from structural models of the transcription elongation complex (EC). Our algorithm is based on the thermodynamic stability of the EC along the DNA template calculated from the sequence-dependent free energy of DNA-DNA, DNA-RNA, and RNA-RNA base pairing associated with (i) the translocational and size fluctuations of the transcription bubble; (ii) changes in the associated DNA-RNA hybrid; and (iii) changes in the cotranscriptional RNA secondary structure upstream of the RNA exit channel. The calculations involve no adjustable parameters except for a cutoff used to discriminate paused from nonpaused complexes. When applied to 100 experimental pauses in transcription elongation by Escherichia coli RNA polymerase on 10 DNA templates, the approach produces statistically significant results. We also present a kinetic model for the rate of recovery of backtracked paused complexes. A crucial ingredient of our model is the incorporation of kinetic barriers to backtracking resulting from steric clashes of EC with the cotranscriptionally generated RNA secondary structure, an aspect not included explicitly in previous attempts at modeling the transcription elongation process.
Vinyard, DJ, Zachary CE, Ananyev GM, Dismukes CG.  2013.  Thermodynamically accurate modeling of the catalytic cycle of photosynthetic oxygen evolution: a mathematical solution to asymmetric Markov chains.. Biochimica et biophysica acta. 1827(7):861-8. Abstract
Forty-three years ago, Kok and coworkers introduced a phenomenological model describing period-four oscillations in O2 flash yields during photosynthetic water oxidation (WOC), which had been first reported by Joliot and coworkers. The original two-parameter Kok model was subsequently extended in its level of complexity to better simulate diverse data sets, including intact cells and isolated PSII-WOCs, but at the expense of introducing physically unrealistic assumptions necessary to enable numerical solutions. To date, analytical solutions have been found only for symmetric Kok models (inefficiencies are equally probable for all intermediates, called "S-states"). However, it is widely accepted that S-state reaction steps are not identical and some are not reversible (by thermodynamic restraints) thereby causing asymmetric cycles. We have developed a mathematically more rigorous foundation that eliminates unphysical assumptions known to be in conflict with experiments and adopts a new experimental constraint on solutions. This new algorithm termed STEAMM for S-state Transition Eigenvalues of Asymmetric Markov Models enables solutions to models having fewer adjustable parameters and uses automated fitting to experimental data sets, yielding higher accuracy and precision than the classic Kok or extended Kok models. This new tool provides a general mathematical framework for analyzing damped oscillations arising from any cycle period using any appropriate Markov model, regardless of symmetry. We illustrate applications of STEAMM that better describe the intrinsic inefficiencies for photon-to-charge conversion within PSII-WOCs that are responsible for damped period-four and period-two oscillations of flash O2 yields across diverse species, while using simpler Markov models free from unrealistic assumptions.
Hudson, BP, Quispe J, Lara-González S, Kim Y, Berman HM, Arnold E, Ebright RH, Lawson CL.  2009.  Three-dimensional EM structure of an intact activator-dependent transcription initiation complex.. Proceedings of the National Academy of Sciences of the United States of America. 106(47):19830-5. Abstract
We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alphaCTD), interactions of alphaCTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.
Lund, G, Prem Das O, Messing J.  1995.  Tissue-specific DNase I-sensitive sites of the maize P gene and their changes upon epimutation. The Plant Journal. 7:797-807.Website
Finelli, AL, Xie T, Bossie CA, Blackman RK, Padgett RW.  1995.  The tolkin gene is a tolloid/BMP-1 homologue that is essential for Drosophila development. Genetics. 141:271-81. AbstractWebsite
The Drosophila decapentaplegic (dpp) gene, a member of the transforming growth factor beta superfamily of growth factors, is critical for specification of the embryonic dorsal-ventral axis, for proper formation of the midgut, and for formation of Drosophila adult structures. The Drosophila tolloid gene has been shown to genetically interact with dpp. The genetic interactions between tolloid and dpp suggests a model in which the tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. We report here the identification and cloning of another Drosophila member of the tolloid/bone morphogenic protein (BMP) 1 family, tolkin, which is located 700 bp 5' to tolloid. Its overall structure is like tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats. Its expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tolkin are expressed uniformly in the imaginal disks. In the brain, both tolloid and tolkin are expressed in the outer proliferation center, whereas tolkin has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tolkin indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp.
Kim, TK, Lagrange T, Wang YH, Griffith JD, Reinberg D, Ebright RH.  1997.  Trajectory of DNA in the RNA polymerase II transcription preinitiation complex.. Proceedings of the National Academy of Sciences of the United States of America. 94(23):12268-73. Abstract
By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, approximately 240 A channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of approximately 50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA.
Padgett, RW, St Johnston RD, Gelbart WM.  1987.  A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-β family. Nature. 325:81-4. AbstractWebsite
The decapentaplegic gene complex (DPP-C) has been implicated in several events in pattern formation during Drosophila development. During embryogenesis, the DPP-C participates in the establishment of dorsal-ventral specification. Later, it is required for the correct morphogenesis of the imaginal disks, which will form much of the adult epidermis. We have undertaken a molecular analysis of the DPP-C to determine what role it plays in positional information. It appears to be a large genetic unit (greater than 40 kilobases (kb] consisting mostly of cis-regulatory information controlling the expression of a set of overlapping transcripts that differ at their 5' ends, but share the bulk of their transcribed sequences. Here, we describe the sequence analysis of two complementary DNAs comprising 4.0 kb of a 4.5-kb transcript. The C-terminus of the protein thereby deduced exhibits strong sequence homology (25-38% amino-acid identity) to the C-termini of a class of mammalian proteins that includes transforming growth factor-beta (TGF-beta), inhibin and Müllerian inhibiting substance (MIS). These proteins act on target cells to produce a variety of responses, such as stimulation or inhibition of cell division or differentiation. The homology suggests that the DPP-C protein contributes to correct morphogenesis as a secreted factor involved in the differential regulation of cell growth. This is the first report of a member of the TGF-beta gene family in a non-mammalian organism, and indicates that one or more members of this gene family existed before arthropod and vertebrate lineages diverged.
Ebright, RH.  1993.  Transcription activation at Class I CAP-dependent promoters.. Molecular microbiology. 8(5):797-802. Abstract
Catabolite gene activator protein (CAP)-dependent promoters can be grouped into three classes, based on the requirement for transcription activation and the position of the DNA site for CAP. Class I CAP-dependent promoters require only CAP for transcription activation and have the DNA site for CAP located upstream of the DNA site for RNA polymerase. Amino acids 156 to 162 of the promoter-proximal subunit of CAP are essential for transcription activation at Class I CAP-dependent promoters, but are not essential for DNA binding, and are not essential for DNA bending. In the structure of the CAP-DNA complex, these amino acids are located in a surface loop and form a cluster on the surface of the CAP-DNA complex. Amino acids 261, 265, and 270 of the alpha subunit of RNA polymerase are essential for response to transcription activation by CAP at Class I CAP-dependent promoters. Several lines of evidence indicate that transcription activation at Class I CAP-dependent promoters requires a direct protein-protein contact between amino acids 156 to 162 of the promoter-proximal subunit of CAP and a molecule of RNA polymerase bound adjacent to CAP on the same face of the DNA helix. It is a strong possibility that this direct protein-protein contact involves amino acids 261 and 265 of the alpha subunit of RNA polymerase.