Li, Y., Segal, G., Wang, Q., Dooner HK.  2013.  Gene tagging with engineered Ds elements in maize. Methods in Molecular Biology: Plant Transposable Elements. :83-99.
Klimuk, E, Akulenko N, Makarova KS, Ceyssens P-J, Lavigne R, Severinov K.  2013.  Host RNA polymerase inhibitors encoded by φKMV-like phages of Pseudomonas. Virology. 436:67-74.
Wu, Y, Yuan L, Guo X, Holding DR, Messing J.  2013.  Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum. Nat Commun. 4:2217. AbstractWebsite
Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a seed storage protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich proteins in the seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.
Guerra, LT, Xu Y, Bennette N, McNeely K, Bryant DA, Dismukes GC.  2013.  Natural osmolytes are much less effective substrates than glycogen for catabolic energy production in the marine cyanobacterium Synechococcus sp. strain PCC 7002 .. J. Biotechnol.. 166:65-75. Abstract
ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.
Barbosa, N, Minakhina S, Medina DJ, Balsara B, Greenwood S, Huzzy L, Rabson AB, Steward R, Schaar DG.  2013.  PDCD2 functions in cancer cell proliferation and predicts relapsed leukemia.. Cancer biology & therapy. 14(6):546-555. AbstractWebsite
PDCD2 is an evolutionarily conserved eukaryotic protein with unknown function. The Drosophlia PDCD2 ortholog Zfrp8 has an essential function in fly hematopoiesis. Zfrp8 mutants exhibit marked lymph gland hyperplasia that results from increased proliferation of partially differentiated hemocytes, suggesting Zfrp8 may participate in cell growth. Based on the above observations we have focused on the role of PDCD2 in human cancer cell proliferation and hypothesized that aberrant PDCD2 expression may be characteristic of human malignancies. We report that PDCD2 is highly expressed in human acute leukemia cells as well as in normal hematopoietic progenitors. PDCD2 knockdown in cancer cells impairs their proliferation, but not viability relative to parental cells, supporting the notion that PDCD2 overexpression facilitates cancer cell growth. Prospective analysis of PDCD2 in acute leukemia patients indicates PDCD2 RNA expression correlates with disease status and is a significant predictor of clinical relapse. PDCD2's role in cell proliferation and its high expression in human malignancies make it an attractive, novel potential molecular target for new anti-cancer therapies.
Guerra, TL, Levitan O, Frada MJ, Sun JS, Falkowski PG, Dismukes GC.  2013.  Regulatory branch points affecting protein and lipid biosynthesis in the diatom Phaeodactylum tricornutum. Biomass and Bioenergy. 59:306-315. AbstractWebsite
It is widely established that nutritional nitrogen deprivation increases lipid accumulation but severely decreases growth rate in microalgae. To understand the regulatory branch points that determine the partitioning of carbon among its potential sinks, we analyzed metabolite and transcript levels of central carbon metabolic pathways and determined the average fluxes and quantum requirements for the synthesis of protein, carbohydrates and fatty acid in the diatom Phaeodactylum tricornutum. Under nitrate-starved conditions, the carbon fluxes into all major sinks decrease sharply; the largest decrease was into proteins and smallest was into lipids. This reduction of carbon flux into lipids together with a significantly lower growth rate is responsible for lower overall FA productivities implying that nitrogen starvation is not a bioenergetically feasible strategy for increasing biodiesel production. The reduction in these fluxes was accompanied by an 18-fold increase in α-ketoglutarate (AKG), 3-fold increase in NADPH/NADP+, and sharp decreases in glutamate (GLU) and glutamine (GLN) levels. Additionally, the mRNA level of acetyl-CoA carboxylase and two type II diacylglycerol-acyltransferases were increased. Partial suppression of nitrate reductase by tungstate resulted in similar trends at lower levels as for nitrate starvation. These results reveal that the GS/GOGAT pathway is the main regulation site for nitrate dependent control of carbon partitioning between protein and lipid biosynthesis, while the AKG/GL(N/U) metabolite ratio is a transcriptional signal, possibly related to redox poise of intermediates in the photosynthetic electron transport system.
Kumaraswamy, GK, Guerra T, Qian X, Zhang S, Bryant DA, Dismukes GC.  2013.  Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: metabolic engineering of NAD+-dependent GAPDH. Energy Environ. Sci.. 6:3722-3731. AbstractWebsite
Catabolism of glycogen stored by cyanobacteria occurs during anaerobic auto-fermentation and produces a range of C1–C3 fermentation products and hydrogen via hydrogenase. We investigated both augmenting and rerouting this carbon catabolism by engineering the glycolysis pathway at the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH-1), its major regulation site at the nexus of two pathways (Oxidative Pentose Phosphate pathway, OPP, and glycolysis/gluconeogenesis). Null (gap1::aphII) and overexpression (gap1+) strains of Synechococcus sp. strain PCC 7002 were constructed in order to produce more NADPH (via rerouting carbon through OPP) and more NADH (via opening the glycolytic bottleneck), respectively. For gap1::aphII quantitative analyses after four days of dark auto-fermentation showed undiminished glycogen catabolism rate, significant increases of intracellular metabolites in both OPP and upper-glycolysis, decrease in lower-glycolysis intermediates, 5.7-fold increase in NADPH, 2.3-fold increase in hydrogen and 1.25-fold increase in CO2vs. wild type (WT). These changes demonstrate the expected outcome of redirection of carbon catabolism through the OPP pathway with significant stimulation of OPP product yields. The gap1+ strain exhibits a large 17% increase in accumulation of glycogen during the prior photoautotrophic growth stage (gluconeogenesis), in parallel with a 2-fold increase in the total [NAD+ + NADH] pool, foreshadowing an increased catabolic capacity. Indeed, the rate of glycogen catabolism during subsequent dark auto-fermentation increased significantly (58%) vs. WT, resulting in increases in both NADH (4.0-fold) and NADPH (2.9-fold) pools, and terminal fermentation products, hydrogen (3.0-fold) D-lactate (2.3-fold) and acetate (1.4-fold). The overall energy conversion yield over four days from catabolized glycogen to hydrogen increased from 0.6 mole of hydrogen per mole of glucose (WT) to 1.4 (gap1::aphII) and 1.1 (gap1+) under headspace accumulation conditions (without hydrogen milking). These findings demonstrate the significant potential of metabolic engineering for redirecting carbon pathways for carbohydrate catabolism and hydrogen production in cyanobacteria.
Gallavotti, A.  2013.  The role of auxin in shaping shoot architecture. Journal of Experimental Botany. 64(9):2593-2608. AbstractWebsite
The variety of plant architectures observed in nature is predominantly determined by vegetative and reproductive branching patterns, the positioning of lateral organs, and differential stem elongation. Branches, lateral organs, and stems are the final products of the activity of meristems, groups of stem cells whose function is genetically deter- mined and environmentally influenced. Several decades of studies in different plant species have shed light on the essential role of the hormone auxin in plant growth and development. Auxin influences stem elongation and regulates the formation, activity, and fate of meristems, and has therefore been recognized as a major hormone shaping plant architecture. Increasing our knowledge of the molecular mechanisms that regulate auxin function is necessary to understand how different plant species integrate a genetically determined developmental programme, the establish- ment of a body plan, with constant inputs from the surrounding environment. This information will allow us to develop the molecular tools needed to modify plant architecture in several crop species and in rapidly changing environments.
Pillitteri LJ, DJ.  2013.  Stomatal development in Arabidopsis.. Arabidopsis Book. :10.1199/tab.0162.
Dooner, HK, Weil CF.  2013.  Transposons and gene creation. Molecular Genetics and Epigenetics of Plant Transposons. :143-167.
Sun, G, Irvine KD.  2013.  Ajuba Family Proteins Link JNK to Hippo Signaling.. Science signaling. 6:ra81. AbstractWebsite
Wounding, apoptosis, or infection can trigger a proliferative response in neighboring cells to replace damaged tissue. Studies in Drosophila have implicated c-Jun amino-terminal kinase (JNK)-dependent activation of Yorkie (Yki) as essential to regeneration-associated growth, as well as growth associated with neoplastic tumors. Yki is a transcriptional coactivator that is inhibited by Hippo signaling, a conserved pathway that regulates growth. We identified a conserved mechanism by which JNK regulated Hippo signaling. Genetic studies in Drosophila identified Jub (also known as Ajuba LIM protein) as required for JNK-mediated activation of Yki and showed that Jub contributed to wing regeneration after wounding and to tumor growth. Biochemical studies revealed that JNK promoted the phosphorylation of Ajuba family proteins in both Drosophila and mammalian cells. Binding studies in mammalian cells indicated that JNK increased binding between the Ajuba family proteins LIMD1 or WTIP and LATS1, a kinase within the Hippo pathway that inhibits the Yki homolog YAP. Moreover, JNK promoted binding of LIMD1 and LATS1 through direct phosphorylation of LIMD1. These results identify Ajuba family proteins as a conserved link between JNK and Hippo signaling, and imply that JNK increases Yki and YAP activity by promoting the binding of Ajuba family proteins to Warts and LATS.
Marcello, MR, Singaravelu G, Singson A.  2013.  Fertilization. Adv. Exp. Med. Biol.. 757:321–350. Abstract
Fertilization-the fusion of gametes to produce a new organism-is the culmination of a multitude of intricately regulated cellular processes. In Caenorhabditis elegans, fertilization is highly efficient. Sperm become fertilization competent after undergoing a maturation process during which they become motile, and the plasma membrane protein composition is reorganized in preparation for interaction with the oocyte. The highly specialized gametes begin their interactions by signaling to one another to ensure that fertilization occurs when they meet. The oocyte releases prostaglandin signals to help guide the sperm to the site of fertilization, and sperm secrete a protein called major sperm protein (MSP) to trigger oocyte maturation and ovulation. Upon meeting one another in the spermatheca, the sperm and oocyte fuse in a specific and tightly regulated process. Recent studies are providing new insights into the molecular basis of this fusion process. After fertilization, the oocyte must quickly transition from the relative quiescence of oogenesis to a phase of rapid development during the cleavage divisions of early embryogenesis. In addition, the fertilized oocyte must prevent other sperm from fusing with it as well as produce an eggshell for protection during external development. This chapter will review the nature and regulation of the various cellular processes of fertilization, including the development of fertilization competence, gamete signaling, sperm-oocyte fusion, the oocyte to embryo transition, and production of an eggshell to protect the developing embryo.
Bao, X, Nickels BE, Fan H.  2012.  Chlamydia trachomatis protein GrgA activates transcription by contacting the nonconserved region of sigma66. Proc Natl Acad Sci U S A. AbstractWebsite
The bacterial RNA polymerase holoenzyme consists of a catalytic core enzyme in complex with a sigma factor that is required for promoter-specific transcription initiation. Primary, or housekeeping, sigma factors are responsible for most of the gene expression that occurs during the exponential phase of growth. Primary sigma factors share four regions of conserved sequence, regions 1-4, which have been further subdivided. Many primary sigma factors also contain a nonconserved region (NCR) located between subregions 1.2 and 2.1, which can vary widely in length. Interactions between the NCR of the primary sigma factor of Escherichia coli, sigma(70), and the beta' subunit of the E. coli core enzyme have been shown to influence gene expression, suggesting that the NCR of primary sigma factors represents a potential target for transcription regulation. Here, we report the identification and characterization of a previously undocumented Chlamydia trachomatis transcription factor, designated GrgA (general regulator of genes A). We demonstrate in vitro that GrgA is a DNA-binding protein that can stimulate transcription from a range of sigma(66)-dependent promoters. We further show that GrgA activates transcription by contacting the NCR of the primary sigma factor of C. trachomatis, sigma(66). Our findings suggest GrgA serves as an important regulator of sigma(66)-dependent transcription in C. trachomatis. Furthermore, because GrgA is present only in chlamydiae, our findings highlight how nonconserved regions of the bacterial RNA polymerase can be targets of regulatory factors that are unique to particular organisms.
Thyssen, G, Svab Z, Maliga P.  2012.  Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny. Plant J.. 72:84-8. AbstractWebsite
Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.
Weiss, LA, Harrison PG, Nickels BE, Glickman MS, Campbell EA, Darst SA, Stallings CL.  2012.  Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis. J Bacteriol. 194:5621-5631. AbstractWebsite
Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP beta subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP beta interactions demonstrates that the CarD/RNAP beta association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP beta interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP beta interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.
Wu, Y, Messing J.  2012.  Rapid divergence of prolamin gene promoters of maize after gene amplification and dispersal. Genetics. 192:507-19. AbstractWebsite
Seeds have evolved to accommodate complicated processes like senescence, dormancy, and germination. Central to these is the storage of carbohydrates and proteins derived from sugars and amino acids synthesized during photosynthesis. In the grasses, the bulk of amino acids is stored in the prolamin superfamily that specifically accumulates in seed endosperm during senescence. Their promoters contain a conserved cis-element, called prolamin-box (P-box), recognized by the trans-activator P-box binding factor (PBF). Because of the lack of null mutants in all grass species, its physiological role in storage-protein gene expression has been elusive. In contrast, a null mutant of another endosperm-specific trans-activator Opaque2 (O2) has been shown to be required for the transcriptional activation of subsets of this superfamily by binding to the O2 box. Here, we used RNAi to knockdown Pbf expression and found that only 27-kDa gamma- and 22-kDa alpha-zein gene expression were affected, whereas the level of other zeins remained unchanged. Still, transgenic seeds had an opaque seed phenotype. Combination of PbfRNAi and o2 resulted in further reduction of alpha-zein expression. We also tested the interaction of promoters and constitutively expressed PBF and O2. Whereas transgenic promoters could be activated, endogenous promoters appeared to be not accessible to transcriptional activation, presumably due to differential chromatin states. Although analysis of the methylation of binding sites of PBF and O2 correlated with the expression of endogenous 22-kDa alpha-zein promoters, a different mechanism seems to apply to the 27-kDa gamma-zein promoter, which does not undergo methylation changes.
Dudas, B, Jenes B, Kiss GB, Maliga P.  2012.  Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in Medicago sativa. Theor. Appl. Genet. 125:1517-23. AbstractWebsite
We report here the isolation of spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective spectinomycin concentrations in the range of 25-50 mg/l. Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2 x Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.
Irvine, KD.  2012.  Integration of intercellular signaling through the Hippo pathway.. Seminars in Cell and Developmental Biology. AbstractWebsite
Metazoan cells are exposed to a multitude of signals, which they integrate to determine appropriate developmental or physiological responses. Although the Hippo pathway was only discovered recently, and our knowledge of Hippo signal transduction is far from complete, a wealth of interconnections amongst Hippo and other signaling pathways have already been identified. Hippo signaling is particularly important for growth control, and I describe how integration of Hippo and other pathways contributes to regulation of organ growth. Molecular links between Hippo signaling and other signal transduction pathways are summarized. Different types of mechanisms for signal integration are described, and examples of how the complex interconnections between pathways are used to guide developmental and physiological growth responses are discussed. Features of Hippo signaling appear to make it particularly well suited to signal integration, including its responsiveness to cell-cell contact and the mediation of its transcriptional output by transcriptional co-activator proteins that can interact with transcription factors of other pathways.
Singaravelu, G, Chatterjee I, Rahimi S, Druzhinina MK, Kang L, Xu XZ, Singson A.  2012.  The sperm surface localization of the TRP-3/SPE-41 Ca2+ -permeable channel depends on SPE-38 function in Caenorhabditis elegans. Dev. Biol.. 365:376–383. Abstract
Despite undergoing normal development and acquiring normal morphology and motility, mutations in spe-38 or trp-3/spe-41 cause identical phenotypes in Caenorhabditis elegans-mutant sperm fail to fertilize oocytes despite direct contact. SPE-38 is a novel, four-pass transmembrane protein and TRP-3/SPE-41 is a Ca(2+)-permeable channel. Localization of both of these proteins is confined to the membranous organelles (MOs) in undifferentiated spermatids. In mature spermatozoa, SPE-38 is localized to the pseudopod and TRP-3/SPE-41 is localized to the whole plasma membrane. Here we show that the dynamic redistribution of TRP-3/SPE-41 from MOs to the plasma membrane is dependent on SPE-38. In spe-38 mutant spermatozoa, TRP-3/SPE-41 is trapped within the MOs and fails to reach the cell surface despite MO fusion with the plasma membrane. Split-ubiquitin yeast-two-hybrid analyses revealed that the cell surface localization of TRP-3/SPE-41 is likely regulated by SPE-38 through a direct protein-protein interaction mechanism. We have identified sequences that influence the physical interaction between SPE-38 and TRP-3/SPE-41, and show that these sequences in SPE-38 are required for fertility in transgenic animals. Despite the mislocalization of TRP-3/SPE-41 in spe-38 mutant spermatozoa, ionomycin or thapsigargin induced influx of Ca(2+) remains unperturbed. This work reveals a new paradigm for the regulated surface localization of a Ca(2+)-permeable channel.
Tungsuchat-Huang, T, Maliga P.  2012.  Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants. Plant J.. 70:717-25. AbstractWebsite
Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.
Ambegaonkar, AA, Pan G, Mani M, Feng Y, Irvine KD.  2012.  Propagation of dachsous-fat planar cell polarity.. Current Biology. 22:1302-1308. AbstractWebsite
The Fat pathway controls both planar cell polarity (PCP) and organ growth [1, 2]. Fat signaling is regulated by the graded expression of the Fat ligand Dachsous (Ds) and the cadherin-domain kinase Four-jointed (Fj). The vectors of these gradients influence PCP [1], whereas their slope can influence growth [3, 4]. The Fj and Ds gradients direct the polarized membrane localization of the myosin Dachs, which is a crucial downstream component of Fat signaling [5-7]. Here we show that repolarization of Dachs by differential expression of Fj or Ds can propagate through the wing disc, which indicates that Fj and Ds gradients can be measured over long range. Through characterization of tagged genomic constructs, we show that Ds and Fat are themselves partially polarized along the endogenous Fj and Ds gradients, providing a mechanism for propagation of PCP within the Fat pathway. We also identify a biochemical mechanism that might contribute to this polarization by showing that Ds is subject to endoproteolytic cleavage and that the relative levels of Ds isoforms are modulated by Fat.
Vvedenskaya, IO, Sharp JS, Goldman SR, Kanabar PN, Livny J, Dove SL, Nickels BE.  2012.  Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs. Genes & development. 26:1498-507. AbstractWebsite
Prokaryotic and eukaryotic RNA polymerases can use 2- to approximately 4-nt RNAs, "nanoRNAs," to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.
Thyssen, G, Svab Z, Maliga P.  2012.  Cell-to-cell movement of plastids in plants. Proc. Natl. Acad. Sci. U.S.A.. 109:2439-43. AbstractWebsite
Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.
Thyssen, G, Svab Z, Maliga P.  2012.  Cell-to-cell movement of plastids in plants. Proceedings of the National Academy of Sciences of the United States of America. 109:2439-43. AbstractWebsite
Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.
Singh, A, Irvine KD.  2012.  Drosophila as a model for understanding development and disease.. Developmental Dynamics. 241:1-2.Website