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.
Xiang, X, Wu Y, Planta J, Messing J, Leustek T.  2017.  Overexpression of serine acetyltransferase in maize leaves increases seed-specific methionine-rich zeins. Plant biotechnology journal. Abstract
Maize kernels do not contain enough of the essential sulphur-amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)-amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S-assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S-assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell-specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12-fold higher SAT activity without negative impact on growth. S-assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10-kDa δ-zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40-fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high-Met zein accumulation. Moreover, SAT overcomes the shortage of S-amino acids that limits the expression and accumulation of high-Met zeins during kernel development.
Yu, L, Winkelman J, Pukhrambam C, Strick T, Nickels BE, Ebright RH.  2017.  The mechanism of variability in transcription start site selection.. eLife. 6:e32038. Abstract
During transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded 'transcription bubble,' and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA 'scrunching' and 'anti-scrunching,' the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.
Garcia, N, Li Y, Dooner HK, Messing J.  2017.  Maize defective kernel mutant generated by insertion of a Ds element in a gene encoding a highly conserved TTI2 cochaperone. Proceedings of the National Academy of Sciences of the United States of America. 114(20):5165-5170. Abstract
We have used the newly engineered transposable element Dsg to tag a gene that gives rise to a defective kernel (dek) phenotype. Dsg requires the autonomous element Ac for transposition. Upon excision, it leaves a short DNA footprint that can create in-frame and frameshift insertions in coding sequences. Therefore, we could create alleles of the tagged gene that confirmed causation of the dek phenotype by the Dsg insertion. The mutation, designated dek38-Dsg, is embryonic lethal, has a defective basal endosperm transfer (BETL) layer, and results in a smaller seed with highly underdeveloped endosperm. The maize dek38 gene encodes a TTI2 (Tel2-interacting protein 2) molecular cochaperone. In yeast and mammals, TTI2 associates with two other cochaperones, TEL2 (Telomere maintenance 2) and TTI1 (Tel2-interacting protein 1), to form the triple T complex that regulates DNA damage response. Therefore, we cloned the maize Tel2 and Tti1 homologs and showed that TEL2 can interact with both TTI1 and TTI2 in yeast two-hybrid assays. The three proteins regulate the cellular levels of phosphatidylinositol 3-kinase-related kinases (PIKKs) and localize to the cytoplasm and the nucleus, consistent with known subcellular locations of PIKKs. dek38-Dsg displays reduced pollen transmission, indicating TTI2's importance in male reproductive cell development.
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.
Walker, SS, Degen D, Nickbarg E, Carr D, Soriano A, Mandal M B, Painter RE, Sheth PR, Xiao L, Sher X et al..  2017.  Affinity selection-mass spectrometry identifies a novel antibacterial RNA polymerase inhibitor.. ACS Chemical Biology. 12:1346-1352. Abstract
The growing prevalence of drug-resistant Gram-negative bacteria is a significant global threat to human health. Rifampicin, an RNA polymerase-targeting agent, is an important part of the antibacterial armamentarium; however the emergence of resistance requires that it be used against only certain infections and usually in combination with another antibiotic. While rifampicin has significant clinical limitations, it does show that bacterial RNA polymerase can be an effective target for antibacterial intervention. To find new RNA polymerase inhibitors we initiated a screen of a collection of antibacterial bioactive molecules using affinity selection-mass spectrometry and purified Escherichia coli core RNA polymerase (subunits α, β, β', ω). Affinity selection screening identified a novel small molecule, MRL-436, that binds selectively to and inhibits RNA polymerase in vitro and inhibits RNA synthesis in the cell. Selection for resistance followed by whole genome sequencing identified a missense mutation in rpoC (β' subunit) and, separately, frameshift mutations in rpoZ (ω subunit) suggesting that MRL-436 targets RNA polymerase in the cell. In addition, cells lacking the rpoZ gene or purified RNA polymerase containing either a specific substitution in β' or lacking ω are selectively resistant to MRL-436. Molecular modeling indicates that the location of the substitution mutation in β' is closely juxtaposed with ω in a region of the complex thought to be important for transcription regulation during cellular response to amino acid starvation.
Lin, W, Mandal S, Degen D, Liu Y, Ebright YW, Li S, Feng Y, Zhang Y, Mandal S, Jiang Y et al..  2017.  Structural basis of Mycobacterium tuberculosis transcription and transcription inhibition.. Molecular Cell. 166:169-179. Abstract
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.
Bird, JG, Nickels BE, Ebright RH.  2017.  RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs.. Bio-protocol. 7(12) Abstract
It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD(+)), reduced nicotinamide adenine dinucleotide (NADH), and 3'-desphospho-coenzyme A (dpCoA), can serve as 'non-canonical initiating nucleotides' (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA polymerases (RNAPs) and that the efficiency of the reaction is determined by promoter sequence (Bird et al., 2016). Here we describe a protocol to quantify the relative efficiencies of transcription initiation using an NCIN vs. transcription initiation using a nucleoside triphosphate (NTP) for a given promoter sequence.
Maffioli, SI, Zhang Y, Degen D, Carzaniga T, Del Gatto G, Serina S, Monciardini P, Mazzetti C, Guglierame P, Candiani G et al..  2017.  Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase.. Cell. 169(7):1240-1248.e23. Abstract
Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy.
Planta, J, Messing J.  2017.  Quality Protein Maize Based on Reducing Sulfur in Leaf Cells. Genetics. 207(4):1687-1697. Abstract
Low levels of the essential amino acids lysine (Lys) and methionine (Met) in a maize-based diet are a major cost to feed and food. Lys deficiency is due to the abundance of Lys-poor proteins in maize kernels. Although a maize mutant, opaque-2 (o2), has sufficient levels of Lys, its soft kernel renders it unfit for storage and transportation. Breeders overcame this problem by selecting quantitative trait loci (QTL) restoring kernel hardness in the presence of o2, a variety called Quality Protein Maize (QPM). Although at least one QTL acts by enhancing the expression of the γ-zein proteins, we could surprisingly achieve rebalancing of the Lys content and a vitreous kernel phenotype by targeting suppression of γ-zeins without the o2 mutant. Reduced levels of γ-zeins were achieved with RNA interference (RNAi). Another transgenic event, PE5 expresses the Escherichia coli enzyme 3'-phosphoadenosine-5'-phosphosulfate reductase involved in sulfate assimilation, specifically in leaves. The stacked transgenic events produce a vitreous endosperm, which has higher Lys level than the classical opaque W64Ao2 variant. Moreover, due to the increased sulfate reduction in the leaf, Met level is elevated in the seed. Such a combination of transgenes produces hybrid seeds superior to classical QPMs that would neither require a costly feed mix nor synthetic Met supplementation, potentially creating a novel and cost-effective means for improving maize nutritional quality.
Garcia, N, Messing J.  2017.  TTT and PIKK Complex Genes Reverted to Single Copy Following Polyploidization and Retain Function Despite Massive Retrotransposition in Maize. Frontiers in plant science. 8:1723. Abstract
The TEL2, TTI1, and TTI2 proteins are co-chaperones for heat shock protein 90 (HSP90) to regulate the protein folding and maturation of phosphatidylinositol 3-kinase-related kinases (PIKKs). Referred to as the TTT complex, the genes that encode them are highly conserved from man to maize. TTT complex and PIKK genes exist mostly as single copy genes in organisms where they have been characterized. Members of this interacting protein network in maize were identified and synteny analyses were performed to study their evolution. Similar to other species, there is only one copy of each of these genes in maize which was due to a loss of the duplicated copy created by ancient allotetraploidy. Moreover, the retained copies of the TTT complex and the PIKK genes tolerated extensive retrotransposon insertion in their introns that resulted in increased gene lengths and gene body methylation, without apparent effect in normal gene expression and function. The results raise an interesting question on whether the reversion to single copy was due to selection against deleterious unbalanced gene duplications between members of the complex as predicted by the gene balance hypothesis, or due to neutral loss of extra copies. Uneven alteration of dosage either by adding extra copies or modulating gene expression of complex members is being proposed as a means to investigate whether the data supports the gene balance hypothesis or not.
Jiao, X, Doamekpor S, Bird JG, Nickels BE, Tong L, Hart RP, Kiledjian M.  2017.  5′-end Nicotinamide Adenine Dinucleotide cap in human cells promotes RNA decay through DXO-mediated deNADding.. Cell. 168(6):1015-1027.
Chatterjee, M, Liu Q, Menello C, Galli M, Gallavotti A.  2017.  The Combined Action of Duplicated Boron Transporters Is Required for Maize Growth in Boron Deficient Conditions. Genetics. 206:2041-2051. AbstractWebsite
The micronutrient boron is essential in maintaining the structure of plant cell walls and is critical for high yields in crop species. Boron can move into plants by diffusion or by active and facilitated transport mechanisms. We recently showed that mutations in the maize boron efflux transporter ROTTEN EAR (RTE) cause severe developmental defects and sterility. RTE is part of a small gene family containing five additional members (RTE2-RTE6) that show tissue specific expression. The close paralogous gene RTE2 encodes a protein with 95% amino acid identity with RTE and is similarly expressed in shoot and root cells surrounding the vasculature. Despite sharing similar functions with RTE, mutations in the RTE2 gene do not cause growth defects in the shoot, even in boron deficient conditions. However, rte2 mutants strongly enhance the rte phenotype in soils with low boron content, producing shorter plants that fail to form all reproductive structures. The joint action of RTE and RTE2 is also required in root development. These defects can be fully complemented by supplying boric acid, suggesting that diffusion or additional transport mechanisms overcome active boron transport deficiencies in the presence of an excess of boron. Overall, these results suggest that RTE2 and RTE function are essential for maize shoot and root growth in boron deficient conditions.
Messing, J.  2017.  Does Investment in Research Always Pay Off? American Academy of Arts and Sciences Bulletin. 70(3):45-47.
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..
Larkins, BA, Wu Y, Song R, Messing J.  2017.  Maize seed storage proteins. Maize Kernel Development. :175-189.
Zhao, C, Wang P, Si T, Hsu C-C, Wang L, Zayed O, Yu Z, Zhu Y, Dong J, Tao AW et al..  2017.  MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability. Developmental Cell. 42:1-12.Website
Bartlett, A, O'Malley R, Huang SC, Galli M, Nery JR, Gallavotti A, Ecker JR.  2017.  Mapping genome-wide transcription factor binding sites using DAP-seq. Nature Protocols. 12(8):1659-1672. AbstractWebsite
To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX). The resulting DNA library is incubated with an affinity-tagged in vitro-expressed TF, and TF–DNA complexes are purified using magnetic separation of the affinity tag. Bound genomic DNA is eluted from the TF and sequenced using next-generation sequencing. Sequence reads are mapped to a reference genome, identifying genome-wide binding locations for each TF assayed, from which sequence motifs can then be derived. A researcher with molecular biology experience should be able to follow this protocol, processing up to 400 samples per week.
Huang, Q, Zhong S, Bleckmann A, Huang J, Guo X, Lin Q, Gu H, Dong J, Dresslhaus T, Qu LJ.  2017.  Sperm cells are passive cargo of the pollen tube in plant fertilization.. Nature Plants. 3:17079
Savage-Dunn, C, Padgett RW.  2017.  The TGF-β Family in Caenorhabditis elegans. The Biology of the TGF-β Family.
Wu, Y, Messing J.  2017.  Understanding and improving protein traits in maize seeds. Achieving Sustainable Maize Cultivation. :115-128.
Gurdon, C, Svab Z, Feng Y, Kumar D, Maliga P.  2016.  Cell-to-cell movement of mitochondria in plants. Proc Natl Acad Sci U S A. 113:3395-400. AbstractWebsite
We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer fromNicotiana sylvestrisintoNicotiana tabacumcells. The alloplasmicN. tabacumline we used carriesNicotiana undulatacytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer fromN. sylvestristoN. tabacumcould be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility toorf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation.
Sun, S, Irvine KD.  2016.  Cellular Organization and Cytoskeletal Regulation of the Hippo Signaling Network.. Trends in cell biology. 26(9):694-704. Abstract
The Hippo signaling network integrates diverse upstream signals to control cell fate decisions and regulate organ growth. Recent studies have provided new insights into the cellular organization of Hippo signaling, its relationship to cell-cell junctions, and how the cytoskeleton modulates Hippo signaling. Cell-cell junctions serve as platforms for Hippo signaling by localizing scaffolding proteins that interact with core components of the pathway. Interactions of Hippo pathway components with cell-cell junctions and the cytoskeleton also suggest potential mechanisms for the regulation of the pathway by cell contact and cell polarity. As our understanding of the complexity of Hippo signaling increases, a future challenge will be to understand how the diverse inputs into the pathway are integrated and to define their respective contributions in vivo.
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.
Pan, Y, Heemskerk I, Ibar C, Shraiman BI, Irvine KD.  2016.  Differential growth triggers mechanical feedback that elevates Hippo signaling.. Proceedings of the National Academy of Sciences of the United States of America. Abstract
Mechanical stress can influence cell proliferation in vitro, but whether it makes a significant contribution to growth control in vivo, and how it is modulated and experienced by cells within developing tissues, has remained unclear. Here we report that differential growth reduces cytoskeletal tension along cell junctions within faster-growing cells. We propose a theoretical model to explain the observed reduction of tension within faster-growing clones, supporting it by computer simulations based on a generalized vertex model. This reduced tension modulates a biomechanical Hippo pathway, decreasing recruitment of Ajuba LIM protein and the Hippo pathway kinase Warts, and decreasing the activity of the growth-promoting transcription factor Yorkie. These observations provide a specific mechanism for a mechanical feedback that contributes to evenly distributed growth, and we show that genetically suppressing mechanical feedback alters patterns of cell proliferation in the developing Drosophila wing. By providing experimental support for the induction of mechanical stress by differential growth, and a molecular mechanism linking this stress to the regulation of growth in developing organs, our results confirm and extend the mechanical feedback hypothesis.