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Blatter, EE, Ross W, Tang H, Gourse RL, Ebright RH.  1994.  Domain organization of RNA polymerase alpha subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding.. Cell. 78(5):889-96. Abstract
Using limited proteolysis, we show that the Escherichia coli RNA polymerase alpha subunit consists of an N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker. We have carried out a detailed structural and functional analysis of an 85 amino acid proteolytic fragment corresponding to the C-terminal domain (alpha CTD-2). Our results establish that alpha CTD-2 has a defined secondary structure (approximately 40% alpha helix, approximately 0% beta sheet). Our results further establish that alpha CTD-2 is a dimer and that alpha CTD-2 exhibits sequence-specific DNA binding activity. Our results suggest a model for the mechanism of involvement of alpha in transcription activation by promoter upstream elements and upstream-binding activator proteins.
Bolot, S, Abrouk M, Masood-Quraishi U, Stein N, Messing J, Feuillet C, Salse J.  2009.  The 'inner circle' of the cereal genomes. Curr Opin Plant Biol. 12:119-25. AbstractWebsite
Early marker-based macrocolinearity studies between the grass genomes led to arranging their chromosomes into concentric 'crop circles' of synteny blocks that initially consisted of 30 rice-independent linkage groups representing the ancestral cereal genome structure. Recently, increased marker density and genome sequencing of several cereal genomes allowed the characterization of intragenomic duplications and their integration with intergenomic colinearity data to identify paleo-duplications and propose a model for the evolution of the grass genomes from a common ancestor. On the basis of these data an 'inner circle' comprising five ancestral chromosomes was defined providing a new reference for the grass chromosomes and new insights into their ancestral relationships and origin, as well as an efficient tool to design cross-genome markers for genetic studies.
Bosacchi, M, Gurdon C, Maliga P.  2015.  Plastid Genotyping Reveals Uniformity of cms-T Maize Cytoplasms.. Plant Physiology. Abstract
Cytoplasmic male sterile (CMS) lines in maize have been classified by their response to specific restorer genes into three categories: cms-C, cms-S, and cms-T. A mitochondrial genome representing each of the CMS cytotypes has been sequenced and male sterility in the cms-S and cms-T cytotypes is linked to chimeric mitochondrial genes. To identify markers for plastid genotyping, we sequenced the plastid genomes (ptDNA) of three fertile maize lines (B37, B73, A188) and the B37 cms-C, cms-S, and cms-T cytoplasmic substitution lines. We found that the plastid genomes of B37 and B73 lines are identical. Furthermore, the fertile and CMS plastid genomes are conserved, differing only by 0-3 single nucleotide polymorphisms (SNPs) in coding regions and 8-22 SNPs and 10-21 short insertions/deletions in noncoding regions. To gain insight into the origin and transmission of the cms-T trait, we identified three SNPs unique to the cms-T plastids, and tested the three diagnostic SNPs in 27 cms-T lines, representing the HA, I, Q, RS and T male sterile cytoplasms. We report that each of the tested 27 cms-T group accessions have the same three diagnostic plastid SNPs indicating a single origin and maternal co-transmission of the cms-T mitochondria and plastids to the seed progeny. Our data exclude exceptional pollen transmission of organelles or multiple horizontal gene transfer events as the source of the urf13-T gene in the cms-T cytoplasms. Plastid genotyping enables a reassessment of evolutionary relationships of cytoplasms in cultivated maize.
Boucher, HW, Ambrose PG, Chambers HF, Ebright RH, Jezek A, Murray BE, Newland JG, Ostrowsky B, Rex JH.  2017.  White Paper: Developing Antimicrobial Drugs for Resistant Pathogens, Narrow-spectrum Indications, and Unmet Needs.. Journal of Infectious Diseases. 216:226-238. Abstract
Despite progress in antimicrobial drug development, a critical need persists for new, feasible pathways to develop antibacterial agents to treat people infected with drug-resistant bacteria. Infections due to resistant Gram-negative bacilli continue to cause unacceptable morbidity and mortality. Antibacterial agents have been traditionally studied in non-inferiority clinical trials that focus on one site of infection (eg, complicated urinary tract infections, intra-abdominal infections), yet these designs may not be optimal, and often are not feasible, for study of infections caused by drug-resistant bacteria. Over the past several years, multiple stakeholders have worked to develop consensus regarding paths forward with a goal of facilitating timely conduct of antimicrobial development. Here we advocate for a novel and pragmatic approach and, towards this end, present feasible trial designs for antibacterial agents that could enable conduct of narrow-spectrum, organism-specific clinical trials and ultimately approval of critically needed new antibacterial agents.
Boyer, LA, Shao X, Ebright RH, Peterson CL.  2000.  Roles of the histone H2A-H2B dimers and the (H3-H4)(2) tetramer in nucleosome remodeling by the SWI-SNF complex.. The Journal of biological chemistry. 275(16):11545-52. Abstract
SWI-SNF is an ATP-dependent chromatin remodeling complex required for expression of a number of yeast genes. Previous studies have suggested that SWI-SNF action may remove or rearrange the histone H2A-H2B dimers or induce a novel alteration in the histone octamer. Here, we have directly tested these and other models by quantifying the remodeling activity of SWI-SNF on arrays of (H3-H4)(2) tetramers, on nucleosomal arrays reconstituted with disulfide-linked histone H3, and on arrays reconstituted with histone H3 derivatives site-specifically modified at residue 110 with the fluorescent probe acetylethylenediamine-(1,5)-naphthol sulfonate. We find that SWI-SNF can remodel (H3-H4)(2) tetramers, although tetramers are poor substrates for SWI-SNF remodeling compared with nucleosomal arrays. SWI-SNF can also remodel nucleosomal arrays that harbor disulfide-linked (H3-H4)(2) tetramers, indicating that SWI-SNF action does not involve an obligatory disruption of the tetramer. Finally, we find that although the fluorescence emission intensity of acetylethylenediamine-(1,5)-naphthol sulfonate-modified histone H3 is sensitive to octamer structure, SWI-SNF action does not alter fluorescence emission intensity. These data suggest that perturbation of the histone octamer is not a requirement or a consequence of ATP-dependent nucleosome remodeling by SWI-SNF.
Bradeen, JM, Timmermans MC, Messing J.  1997.  Dynamic genome organization and gene evolution by positive selection in geminivirus (Geminiviridae). Molecular biology and evolution. 14:1114-24. AbstractWebsite
Geminiviruses (Geminiviridae) are a diverse group of plant viruses differing from other known plant viruses in possessing circular, single-stranded DNA. Current classification divides the family into three subgroups, defined in part by genome organization, insect vector, and plant host range. Previous phylogenetic assessments of geminiviruses have used DNA and/or amino acid sequences from the replication-associated and coat protein genes and have relied predominantly on distance analyses. We used amino acid and DNA sequence data from the replication-associated and coat protein genes from 22 geminivirus types in distance and parsimony analyses. Although the results of our analyses largely agree with those reported previously, we could not always predict viral relationships based on genome organization, plant host, or insect vector. Loss of correlation of these traits with phylogeny is likely due to improved sampling of geminivirus types. Unrooted parsimony trees suggest multiple independent origins for the monopartite genome. genome organization is therefore a dynamic character. Estimates of nonsynonymous and synonymous nucleotide substitutions for extant and inferred ancestral sequences were used to evaluate hypotheses that the replication-associated and coat protein sequences evolve to accommodate plant host and insect vector specificities, respectively. Results suggest that plant host specificity does not solely direct replication-associated protein-evolution but that coat protein sequence does evolve in response to insect vector specificity. Genome organization and, possibly, plant host specificity are not reliable taxonomic characters.
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.
Brimblecombe, R, Koo A, Dismukes CG, Swiegers GF, Spiccia L.  2010.  Solar Driven Water Oxidation by a Bioinspired Manganese Molecular Catalyst. Journal of the American Chemical Society. 132:2892-2894. AbstractWebsite
Brimblecombe, R, Dismukes CG, Swiegers GF, Spiccia L.  2009.  Molecular water-oxidation catalysts for photoelectrochemical cells. Dalton Transactions. :9374-9384. AbstractWebsite
Photoelectrochemical cells that efficiently split water into oxygen and hydrogen, "the fuel of the future", need to combine robust water oxidation catalysts at the anode (2H2O [rightward arrow] O2 + 4H+ + 4e-) with hydrogen reduction catalysts at the cathode (2H+ + 2e-[rightward arrow] H2). Both sets of catalysts will, ideally, operate at low overpotentials and employ light-driven or light-assisted processes. In this Perspective article, we focus on significant efforts to develop solid state materials and molecular coordination complexes as catalyst for water oxidation. We briefly review the field with emphasis on the various molecular catalysts that have been developed and then examine the activity of molecular catalysts in water oxidation following their attachment to conducting electrodes. For such molecular species to be useful in a solar water-splitting device it is preferable that they are securely and durably affixed to an electrode surface. We also consider recent developments aimed at combining the action of molecular catalysts with light absorption so that light driven water oxidation may be achieved.
Brimblecombe, R, Swiegers G F, Dismukes  CG, Spiccia L.  2008.  Sustained Water Oxidation Photocatalysis by a Bioinspired Manganese Cluster. Angewandte Chemie International Edition. 47:7335-7338.Website
Brimblecombe, R, Kolling DRJ, Bond AM, Dismukes CG, Swiegers GF, Spiccia L.  2009.  Sustained Water Oxidation by [Mn4O4]7+ Core Complexes Inspired by Oxygenic Photosynthesis. Inorganic Chemistry. 48:7269-7279. AbstractWebsite
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
Brimblecombe, R, Chen J, Wagner P, Buchhorn T, Dismukes CG, Spiccia L, Swiegers GF.  2011.  Photocatalytic oxygen evolution from non-potable water by a bioinspired molecular water oxidation catalyst. Journal of Molecular Catalysis A: Chemical. 338:1-6.Website
Brown, BA, Padgett RW, Hardies SC, Hutchison CA, Edgell MH.  1982.  β-globin transcript found in induced murine erythroleukemia cells is homologous to the beta h0 and beta h1 genes. Proceedings of the National Academy of Sciences of the United States of America. 79:2753-7. AbstractWebsite
RNA transcripts complementary to at least one of the four beta-globin homologous genes (beta h) are found in high concentration in the murine erythroleukemic (MEL) cell line GM979 after butyric acid induction. Hybridization data indicate that the gene expressed is Hbb-beta h0 or Hbb-beta h1, or both. The level of beta h0/1 transcripts in the MEL cell is similar to the level of adult transcripts. The Hbb-beta h0/1 transcript is about 800 nucleotides in length. In addition, there is a larger beta h0/1 transcript of the same size and relative intensity as the adult beta-globin precursor. We also report significant levels of embryonic gene Hbb-y transcripts in induced GM979 cells. We have determined that the GM979 cell line has the [Hbb]s haplotype on the basis of an examination of its globin DNA restriction pattern. An additional MEL cell line of haplotype [Hbb]d (DBA/2 line 6A11A) was examined and found to contain no significant level of Hbb-beta h0, Hbb-beta h1, Hbb-beta h2, or Hbb-y gene transcripts.
Bruggmann, R, Bharti AK, Gundlach H, Lai J, Young S, Pontaroli AC, Wei F, Haberer G, Fuks G, Du C et al..  2006.  Uneven chromosome contraction and expansion in the maize genome. Genome research. 16:1241-51. AbstractWebsite
Maize (Zea mays or corn), both a major food source and an important cytogenetic model, evolved from a tetraploid that arose about 4.8 million years ago (Mya). As a result, maize has extensive duplicated regions within its genome. We have sequenced the two copies of one such region, generating 7.8 Mb of sequence spanning 17.4 cM of the short arm of chromosome 1 and 6.6 Mb (25.6 cM) from the long arm of chromosome 9. Rice, which did not undergo a similar whole genome duplication event, has only one orthologous region (4.9 Mb) on the short arm of chromosome 3, and can be used as reference for the maize homoeologous regions. Alignment of the three regions allowed identification of syntenic blocks, and indicated that the maize regions have undergone differential contraction in genic and intergenic regions and expansion by the insertion of retrotransposable elements. Approximately 9% of the predicted genes in each duplicated region are completely missing in the rice genome, and almost 20% have moved to other genomic locations. Predicted genes within these regions tend to be larger in maize than in rice, primarily because of the presence of predicted genes in maize with larger introns. Interestingly, the general gene methylation patterns in the maize homoeologous regions do not appear to have changed with contraction or expansion of their chromosomes. In addition, no differences in methylation of single genes and tandemly repeated gene copies have been detected. These results, therefore, provide new insights into the diploidization of polyploid species.
Burke, JM, Irvine KD, Kaneko KJ, Kerker BJ, Oettgen AB, Tierney WM, Williamson CL, Zaug AJ, Cech TR.  1986.  Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor. Cell. 45:167-76. AbstractWebsite
Oligonucleotide-directed mutagenesis has been used to alter highly conserved sequences within the intervening sequence (IVS) of the Tetrahymena large ribosomal RNA precursor. Mutations within either sequence element 9L or element 2 eliminate splicing activity under standard in vitro splicing conditions. A double mutant with compensatory base changes in elements 9L and 2 has accurate splicing activity restored. Thus, the targeted nucleotides of elements 9L and 2 base-pair with one another in the IVS RNA, and pairing is important for self-splicing. Mutant splicing activities are restored by increased magnesium ion concentrations, supporting the conclusion that the role of the targeted bases in splicing is primarily structural. Based on the temperature dependence, we propose that a conformational switch involving pairing and unpairing of elements 9L and 2 is required for splicing.
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.
Busby, S, Ebright RH.  1999.  Transcription activation by catabolite activator protein (CAP).. Journal of molecular biology. 293(2):199-213. Abstract
Transcription activation by Escherichia coli catabolite activator protein (CAP) at each of two classes of simple CAP-dependent promoters is understood in structural and mechanistic detail. At class I CAP-dependent promoters, CAP activates transcription from a DNA site located upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at these promoters, transcription activation involves protein-protein interactions between CAP and the RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex. At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex; and (ii) protein-protein interactions between CAP and RNAP alpha subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open complex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits synergistic transcription activation by multiple molecules of CAP, or by CAP and other activators. Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits "anti-activation" by negative regulators. Basic features of transcription activation at class I and class II CAP-dependent promoters appear to be generalizable to other activators.
Busby, S, Ebright RH.  1997.  Transcription activation at class II CAP-dependent promoters.. Molecular microbiology. 23(5):853-9. Abstract
Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how a single activator molecule can make multiple interactions with the transcription machinery, with each interaction being responsible for a specific mechanistic consequence. At Class II CAP-dependent promoters, the DNA target site for CAP is centred near position -42, overlapping and replacing the -35 determinant for binding of RNA polymerase (RNAP). Transcription activation requires two distinct mechanistic components. The first component is 'anti-inhibition,' overcoming an inhibitory effect of the RNAP alpha subunit C-terminal domain (alpha CTD). This component involves direct contact between amino acids 156-164 (activating region 1) of the upstream subunit of the CAP dimer and a target in alpha CTD. The second component is 'direct activation', facilitating isomerization of the RNAP-promoter closed complex to the transcriptionally competent open complex. This component involves direct contact between amino acids 19, 21 and 101 (activating region 2) of the downstream subunit of the CAP dimer and a target in the RNAP alpha subunit N-terminal domain (alpha NTD).
Calvino, M., Bruggmann R, Messing J.  2011.  Characterization of the small RNA component of the transcriptome from grain and sweet sorghum stems. BMC Genomics. 12:356. AbstractWebsite
ABSTRACT: BACKGROUND: Sorghum belongs to the tribe of the Andropogoneae that includes potential biofuel crops like switchgrass, Miscanthus and successful biofuel crops like corn and sugarcane. However, from a genomics point of view sorghum has compared to these other species a simpler genome because it lacks the additional rounds of whole genome duplication events. Therefore, it has become possible to generate a high-quality genome sequence. Furthermore, cultivars exists that rival sugarcane in levels of stem sugar so that a genetic approach can be used to investigate which genes are differentially expressed to achieve high levels of stem sugar. RESULTS: Here, we characterized the small RNA component of the transcriptome from grain and sweet sorghum stems, and from F2 plants derived from their cross that segregated for sugar content and flowering time. We found that variation in miR172 and miR395 expression correlated with flowering time whereas variation in miR169 expression correlated with sugar content in stems. Interestingly, genotypic differences in the ratio of miR395 to miR395* were identified, with miR395* species expressed as abundantly as miR395 in sweet sorghum but not in grain sorghum. Finally, we provided experimental evidence for previously annotated miRNAs detecting the expression of 25 miRNA families from the 27 known and discovered 9 new miRNAs candidates in the sorghum genome. CONCLUSIONS: Sequencing the small RNA component of sorghum stem tissue provides us with experimental evidence for previously predicted microRNAs in the sorghum genome and microRNAs with a potential role in stem sugar accumulation and flowering time.
Calvino, M., Messing J.  2013.  Discovery of MicroRNA169 gene copies in genomes of flowering plants through positional information. Genome Biol Evol. 5:402-17. AbstractWebsite
Expansion and contraction of microRNA (miRNA) families can be studied in sequenced plant genomes through sequence alignments. Here, we focused on miR169 in sorghum because of its implications in drought tolerance and stem-sugar content. We were able to discover many miR169 copies that have escaped standard genome annotation methods. A new miR169 cluster was found on sorghum chromosome 1. This cluster is composed of the previously annotated sbi-MIR169o together with two newly found MIR169 copies, named sbi-MIR169t and sbi-MIR169u. We also found that a miR169 cluster on sorghum chr7 consisting of sbi-MIR169l, sbi-MIR169m, and sbi-MIR169n is contained within a chromosomal inversion of at least 500 kb that occurred in sorghum relative to Brachypodium, rice, foxtail millet, and maize. Surprisingly, synteny of chromosomal segments containing MIR169 copies with linked bHLH and CONSTANS-LIKE genes extended from Brachypodium to dictotyledonous species such as grapevine, soybean, and cassava, indicating a strong conservation of linkages of certain flowering and/or plant height genes and microRNAs, which may explain linkage drag of drought and flowering traits and would have consequences for breeding new varieties. Furthermore, alignment of rice and sorghum orthologous regions revealed the presence of two additional miR169 gene copies (miR169r and miR169s) on sorghum chr7 that formed an antisense miRNA gene pair. Both copies are expressed and target different set of genes. Synteny-based analysis of microRNAs among different plant species should lead to the discovery of new microRNAs in general and contribute to our understanding of their evolution.
Calviño, M, Messing J.  2012.  Sweet sorghum as a model system for bioenergy crops.. Current opinion in biotechnology. 23(3):323-9. AbstractWebsite
Bioenergy is the reduction of carbon via photosynthesis. Currently, this energy is harvested as liquid fuel through fermentation. A major concern, however, is input cost, in particular use of excess water and nitrogen, derived from an energy-negative process, the Haber-Bosch method. Furthermore, the shortage of arable land creates competition between uses for food and fuel, resulting in increased living expenses. This review seeks to summarize recent knowledge in genetics, genomics, and gene expression of a rising model species for bioenergy applications, sorghum. Its diploid genome has been sequenced, it has favorable low-input cost traits, and genetic crosses between different cultivars can be used to study allelic variations of genes involved in stem sugar metabolism and incremental biomass.