Xu, J-H, Liu Q, Hu W, Wang T, Xue Q, Messing J.  2015.  Dynamics of chloroplast genomes in green plants.. Genomics. 106(4):221-31. Abstract
Chloroplasts are essential organelles, in which genes have widely been used in the phylogenetic analysis of green plants. Here, we took advantage of the breadth of plastid genomes (cpDNAs) sequenced species to investigate their dynamic changes. Our study showed that gene rearrangements occurred more frequently in the cpDNAs of green algae than in land plants. Phylogenetic trees were generated using 55 conserved protein-coding genes including 33 genes for photosynthesis, 16 ribosomal protein genes and 6 other genes, which supported the monophyletic evolution of vascular plants, land plants, seed plants, and angiosperms. Moreover, we could show that seed plants were more closely related to bryophytes rather than pteridophytes. Furthermore, the substitution rate for cpDNA genes was calculated to be 3.3×10(-10), which was almost 10 times lower than genes of nuclear genomes, probably because of the plastid homologous recombination machinery.
Zhang, W, Garcia N, Feng Y, Zhao H, Messing J.  2015.  Genome-wide histone acetylation correlates with active transcription in maize.. Genomics. 106(4):214-20. Abstract
Gene expression is regulated at many different levels during the life cycle of all plant species. Recent investigations have taken advantage of next-generation sequencing to study the relevance of DNA methylation and sRNAs in controlling tissue-specific gene expression in maize at the genome-wide level. Here, we profiled H3K27ac in maize, which has one of the largest sequenced plant genomes due to the amplification of retrotransposons. Because transcribed genes represent only a small proportion of its genome, gene-specific epigenetic modifications are concentrated in a relatively small percentage of the genome. Indeed, H3K27ac marks are mostly in gene-rich, in contrast to gene-poor regions. A large proportion of those marks are located in transcribed regions of genes, including 111 out of 458 known genetic loci. Moreover, increased transcription correlates with the presence of H3K27ac modification in gene bodies. Using maize as an example, we suggest that H3K27ac marks actively transcribed genes in plants.
Vvedenskaya, IO, Zhang Y, Goldman SR, Valenti A, Visone V, Taylor DM, Ebright RH, Nickels BE.  2015.  Massively Systematic Transcript End Readout, "MASTER": Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields.. Molecular cell. 60:953-965. Abstract
We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 4(7) (∼16,000) barcoded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields (massively systematic transcript end readout, "MASTER"). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo; we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching").
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.
Hassan, HM, Degen D, Jang K H, Ebright RH, Fenical W.  2015.  Salinamide F, new depsipeptide antibiotic and inhibitor of bacterial RNA polymerase from a marine-derived Streptomyces sp.. The Journal of antibiotics. 68(3):206-9.
Feng, Y, Degen D, Wang X, Gigliotti M, Liu S, Zhang Y, Das D, Michalchuk T, Ebright YW, Talaue M et al..  2015.  Structural basis of transcription inhibition by CBR hydroxamidines and CBR pyrazoles. Structure. 23:1470-1481. AbstractWebsite
CBR hydroxamidines are small-molecule inhibitors of bacterial RNA polymerase (RNAP) discovered through high-throughput screening of synthetic-compound libraries. CBR pyrazoles are structurally related RNAP inhibitors discovered through scaffold hopping from CBR hydroxamidines. CBR hydroxamidines and pyrazoles selectively inhibit Gram-negative bacterial RNAP and exhibit selective antibacterial activity against Gram-negative bacteria. Here, we report crystal structures of the prototype CBR hydroxamidine, CBR703, and a CBR pyrazole in complex with E. coli RNAP holoenzyme. In addition, we define the full resistance determinant for CBR703, show that the binding site and resistance determinant for CBR703 do not overlap the binding sites and resistance determinants of other characterized RNAP inhibitors, show that CBR703 exhibits no or minimal cross-resistance with other characterized RNAP inhibitors, and show that co-administration of CBR703 with other RNAP inhibitors results in additive antibacterial activities. The results set the stage for structure-based optimization of CBR inhibitors as antibacterial drugs.
Głuszek, AA, Cullen FC, Li W, Battaglia RA, Radford SJ, Costa MF, McKim KS, Goshima G, Ohkura H.  2015.  The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes.. The Journal of cell biology. 211(6):1113-20. Abstract
The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore-microtubule attachment in oocytes.
Liu, Z, Li X, Wang T, Messing J, Xu J-H.  2015.  The Wukong Terminal-Repeat Retrotransposon in Miniature (TRIM) Elements in Diverse Maize Germplasm.. G3 (Bethesda, Md.). 5(8):1585-92. AbstractWebsite
TRIMs (terminal-repeat retrotransposons in miniature), which are characterized by their small size, have been discovered in all investigated vascular plants and even in animals. Here, we identified a highly conservative TRIM family referred to as Wukong elements in the maize genome. The Wukong family shows a distinct pattern of tandem arrangement in the maize genome suggesting a high rate of unequal crossing over. Estimation of insertion times implies a burst of retrotransposition activity of the Wukong family after the allotetraploidization of maize. Using next-generation sequencing data, we detected 87 new Wukong insertions in parents of the maize NAM population relative to the B73 reference genome and found abundant insertion polymorphism of Wukong elements in 75 re-sequenced maize lines, including teosinte, landraces, and improved lines. These results suggest that Wukong elements possessed a persistent retrotransposition activity throughout maize evolution. Moreover, the phylogenetic relationships among 76 maize inbreds and their relatives based on insertion polymorphisms of Wukong elements should provide us with reliable molecular markers for biodiversity and genetics studies.
Wang, W, Zhang W, Wu Y, Maliga P, Messing J.  2015.  RNA Editing in Chloroplasts of Spirodela polyrhiza, an Aquatic Monocotelydonous Species.. PloS one. 10(10):e0140285. Abstract
RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor.
Chakraborty, A, Mazumder A, Lin M, Hasemeyer A, Xu Q, Wang D, Ebright YW, Ebright RH.  2015.  Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.. Methods in molecular biology (Clifton, N.J.). 1276:101-31. Abstract
A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.
Galli, M, Liu Q, Moss BL, Malcomber S, Li W, Gaines C, Federici S, Roshkovan J, Meeley R, Nemhauser J et al..  2015.  Auxin signaling modules regulate maize inflorescence architecture. Proc Natl Acad Sci USA. 112:13372-13377. AbstractWebsite
In plants, small groups of pluripotent stem cells called axillary meristems are required for the formation of the branches and flowers that eventually establish shoot architecture and drive reproductive success. To ensure the proper formation of new axillary meristems, the specification of boundary regions is required for coordinating their development. We have identified two maize genes, BARREN INFLORESCENCE1 and BARREN INFLORESCENCE4 (BIF1 and BIF4), that regulate the early steps required for inflorescence formation. BIF1 and BIF4 encode AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, which are key components of the auxin hormone signaling pathway that is essential for organogenesis. Here we show that BIF1 and BIF4 are integral to auxin signaling modules that dynamically regulate the expression of BARREN STALK1 (BA1), a basic helix-loop-helix (bHLH) transcriptional regulator necessary for axillary meristem formation that shows a striking boundary expression pattern. These findings suggest that auxin signaling directly controls boundary domains during axillary meristem formation and define a fundamental mechanism that regulates inflorescence architecture in one of the most widely grown crop species.
Zhang, Y, Wang P, Shao W, Zhu J-K, Dong J.  2015.  The BASL Polarity Protein Controls a MAPK Signaling Feedback Loop in Asymmetric Cell Division.. Dev Cell. doi: 10.1016/j.devcel.2015.02.022.
Druzhinin, SY, Tran NT, Skalenko KS, Goldman SR, Knoblauch JG, Dove SL, Nickels BE.  2015.  A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq. PLoS Genet. 11(7):e1005348.
Ambegaonkar, AA, Irvine KD.  2015.  Coordination of planar cell polarity pathways through Spiny legs. eLife. 4:pii:e09946.
Singaravelu, G, Rahimi S, Krauchunas A, Rizvi A, Dharia S, Shakes D, Smith H, Golden A, Singson A.  2015.  Forward Genetics Identifies a Requirement for the Izumo-like Immunoglobulin Superfamily spe-45 Gene in Caenorhabditis elegans Fertilization.. Current Biology. 25:3220-3224.
Cruz, JW, Sharp JD, Hoffer ED, Maehigashi T, Vvedenskaya IO, Konkimalla A, Husson RN, Nickels BE, Dunham C, Woychik NA.  2015.  Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase. Nat Commun. 6:7480.
Qian, X, Kumaraswamy GK, Zhang S, Gates C, Ananyev GM, Bryant DA, Dismukes GC.  2015.  Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.. Biotechnol Bioeng. 113(5):979-988. Abstract
To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations-both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))-were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.
Radford, SJ, Hoang TL, Gluszek AA, Ohkura H, McKim KS.  2015.  Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes. PLoS Genetics. 11(10):e1005605.Website
Vvedenskaya, IO, Zhang Y, Goldman SR, Valenti A, Visone V, Taylor DM, Ebright RH, Nickels BE.  2015.  Massively systematic transcript end readout, “MASTER”: transcription start site selection, transcriptional slippage, and transcript yields. Molecular Cell. 60:953-965.
Akhurst, RJ, Padgett RW.  2015.  Matters of context guide future research in TGFβ superfamily signaling. Science Signaling. 8(399):DOI:10.1126/scisignal.aad0416.
Krishnan, A, Kumaraswamy GK, Vinyard DJ, Gu H, Ananyev GM, Posewitz MZ, Dismukes GC.  2015.  Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant.. Plant J. 81(6):947-960. Abstract
Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.
Gallavotti, A. and Whipple, CJ.  2015.  Positional cloning in maize (Zea mays subsp. mays, Poaceae). Applications in Plant Sciences. 3:1400092.Website
Vvedenskaya, IO, Goldman SR, Nickels BE.  2015.  Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5' ends. Methods Mol Biol. 1276:211-228.
Goldman, SR, Nair N, Wells C, Nickels BE, Hochschild A.  2015.  The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo. eLife. 4:e10514.