Robustness and Recoverability in Transport Logistics

Abstract Transport is the foundation of any economy: it boosts economic growth, creates wealth, enhances trade, geographical accessibility and the mobility of people. Transport is also a key ingredient for a high quality of life, making places accessible and bringing people together. The future prosperity of our world will depend on the ability of all of its regions to remain fully and competitively integrated in the world economy. Efficient transport is vital in making this happen. Operations research can help in efficiently planning the design and operating transport systems. Planning and operational processes are fields that are rich in combinatorial optimization problems. These problems can be analyzed and solved through the application of mathematical models and optimization techniques, which may lead to an improvement in the performance of the transport system, as well as to a reduction in the time required for solving these problems. The latter aspect is important, because it increases the flexibility of the system: the system can adapt in a faster way to changes in the environment (i.e.: weather conditions, crew illness, failures, etc.). These disturbing changes (called disruptions) often enforce the schedule to be adapted. The direct consequences are delays and cancellations, implying many schedule adjustments and huge costs. Consequently, robust schedules and recovery plans must be developed in order to fight against disruptions. This dissertation makes contributions to two different fields: rail and air applications. Robust planning and recovery methods are presented. In the field of railway transport we develop several mathematical models which answer to RENFE’s (the major railway operator in Spain) needs: 1. We study the rolling stock assignment problem: here, we introduce some robust aspects in order to ameliorate some operations which are likely to fail. Once the rolling stock assignment is known, we propose a robust routing model which aims at identifying the train units’ sequences while minimizing the expected delays and human resources needed to perform the sequences. 2. It is widely accepted that the sequential solving approach produces solutions that are not global optima. Therefore, we develop an integrated and robust model to determine the train schedule and rolling stock assignment. We also propose an integrated model to study the rolling stock circulations. Circulations are determined by the rolling stock assignment and routing of the train units. 3. Although our aim is to develop robust plans, disruptions will be likely to occur and recovery methods will be needed. Therefore, we propose a recovery method which aims to recover the train schedule and rolling stock assignment in an integrated fashion all while considering the passenger demand. In the field of air transport we develop several mathematical models which answer to IBERIA’s (the major airline in Spain) needs: 1. We look at the airline-scheduling problem and develop an integrated approach that optimizes schedule design, fleet assignment and passenger use so as to reduce costs and create fewer incompatibilities between decisions. Robust itineraries are created to ameliorate misconnected passengers. 2. Air transport operators are continuously facing competition from other air operators and different modes of transport (e.g., High Speed Rail). Consequently, airline profitability is critically influenced by the airline’s ability to estimate passenger demands and construct profitable flight schedules. We consider multi-modal competition including airline and rail, and develop a new approach that estimates the demand associated with a given schedule; and generates airline schedules and fleet assignments using an integrated schedule design and fleet assignment optimization model that captures the impacts of schedule decisions on passenger demand.

Tecnología y competitividad en la economía mundial. Factores e indicadores clave de actuación

Durante los últimos 30 años se han creado una gran cantidad de índices e indicadores para evaluar la práctica totalidad de los países bajo distintas premisas. La tesis parte de un análisis detallado de más de un centenar de estos índices diferenciados entre los enfocados al desarrollo y los enfocados a la competitividad económica (véase Módulo I anexo) y tras esto, dentro del estudio teórico nos hemos centrado en 35 indicadores relacionados con la tecnología (capítulo 3, apartado 3.3.). La justificación, el objetivo de la investigación y la estructura de la tesis se presenta en el capítulo 2. Respecto a la metodología, tal y como se plantea en la hipótesis (apartado 2.2.), se presentan los criterios de selección de seis grupos de países (EP, EPC, EC, ECI, EI y EM)1, que se van a evaluar. Posteriormente se plantea el Protocolo de Cálculo para el total de grupos seleccionados dentro de los periodos 2005-2006 y 2007-2008 y se realiza una profunda evaluación estadística como se plantea dentro de la coherencia estadística explicada en el apartado 3.4.4.5. (también se dispone de los cálculos dentro de los Módulos II, III, IV y V anexos). Tras la metodología establecemos la construcción de un índice sintético NRI(A) y, tras esto, estudiamos las relaciones así como la interpretación de los resultados (capítulo 4, apartado 4.1.). Una vez obtenidos los resultados realizamos la validación de los mismos para el periodo 2007-2015 (capítulo 4 - apartado 4.2. – y los Módulos VI y VII anexos). En el capítulo 5, evaluamos el nivel de preparación tecnológico y su relación con la competitividad para los seis grupos de países (véase desde los apartados 5.1.y 5.2.). Dentro de cada uno de los seis grupos de países, sabemos las variables que cualitativamente tienen que priorizarse para mejorar el nivel de preparación tecnológica de los mismos. Estas variables inicialmente son sesenta y ocho – año 2007-08 –, y al final de la implementación del método se reducen notablemente. Estas variables finales, llamadas Indicadores Clave de Actuación (ICA), se agrupan – vía análisis factorial – en Factores Clave de Actuación que nos simplifican lo planteado. Para cada grupo de países se realizan los conglomerados de acuerdo a sus valores dentro de las ICAs en busca de singularidades y se ha llevado a cabo un análisis minucioso en función de los Indicadores Clave de Actuación. La Tesis, plantea científicamente como podemos evaluar el nivel de preparación tecnológica y su relación con la competitividad, desde un índice sintético creado NRI(A), que contempla únicamente Indicadores Clave de Actuación (variables seleccionadas) a partir de las variables originales del Network Readiness Index (NRI(R)) . Por último se plantea dentro de las conclusiones, capítulo 6, diferentes líneas de investigación, desarrollando dos de ellas que se pueden encontrar en el Modulo VIII anexo. Por un lado presentamos una línea de investigación centrada en 29 economías africanas (EA) de las que disponemos información fidedigna y por otro lado una segunda línea en la que nos centramos en la evaluación de España respecto a sus naciones coetáneas. La principal voluntad de la presente tesis doctoral, es simplificar la evaluación del nivel de preparación tecnológica y la relación de esta con la competitividad a partir de la creación de un índice sintético propio NRI(A). ABSTRACT - During the last 30 years, many institutions have been evaluating and endless range of variables in practically all of the world´s economies. This Thesis is the product of a detail analysis of more than one hundred indicators / index, which we have divided into two parts: those focused on development and those focused on economic competitiveness (see module I annex). Secondly, in our theoretical research we have concentrated on those indicators, which are related to technology (chapters 3, section 3.3). The selection criteria of the six economic groups to be evaluated are included in our methodology, as mentioned in the hypothesis (see section 2.2.). Subsequently the calculation procedure is also presented for all of the groups selected between the periods 2005-2006 and 2007-2008. Next, we perform a statistical study, which is presented accordingly in the segment dealing with statistics, section 3.4.4.5. The calculations are provided in modules I, II, III, IV and V annex. After the methods segment of the Thesis, we develop our argument, in which we presented the explanation of the relations as well as the interpretation of the results. Also at the chapter 4 you can find the result validation from 2007 till 2015. Finally in chapters 6, we evaluate the conclusions for the six economic groups (see section 6.2.). The Thesis scientifically explains the way in which we evaluate economic competitiveness in 135 countries from a standpoint of strictly technological variables. Six groups of countries are evaluated, being divided by criteria, which homogenize the economies under review. We recognize that the variables of each economic group should be prioritized in order to better their competitiveness. Initially the group consisted of 68 variables, a number which was considerably reduced after the implementation of our methodology. Likewise, these final variables, dubbed “key performance indicators”, were grouped into factors (key performance factors), which greatly simplify the prioritization process. At the same time, conglomerates have been created for each economic group according to their value concerning the selected variables. A detailed country – by – country analysis of their positioning in each of the six groups was conducted for each of the mathematically selected key performance indicators. Finally, at the Conclusion we introduce new research lines and between them we focus on two research lines in which ones we are working with (see chapter 6). We basically try to apply the multivariable analysis method, the factorial analysis and the conglomerates to designed and implemented our method first in a geographically group of countries (Africa) and secondly to evaluate and develop the public policies for Spain for the development of its competitively, comparing Spain to his coetaneous countries in Europe (see Module VIII). The main objective of this Doctoral Thesis is to noticeably simplify the comparison of the Network Readiness Index and its relation with the economic competitiveness of the countries using a new synthetic index design by us.

Transfer of human serum IgG to nonobese diabetic Igμnull mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren’s syndrome

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sjögren’s syndrome. NOD.Igμnull mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igμnull mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab′)2 fragments from parental NOD mice or human primary Sjögren’s syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sjögren’s syndrome.

Structural origins of the exonuclease resistance of a zwitterionic RNA

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2′-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1°C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3′ ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3′-5′ exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-Å resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 Å and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2′-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.

Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I

N-type Ca2+ channels mediate Ca2+ influx, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca2+ channels with synaptotagmin I, an important Ca2+ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca2+. Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca2+-dependent because of the Ca2+ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca2+ channels and synaptotagmin, and suggest that N-type Ca2+ channels may undergo a complex series of Ca2+-dependent interactions with multiple presynaptic proteins during neurotransmission.

Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: Regulation of HSP90-binding activity of FKBP52

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.

Signal Recognition Particle-dependent Targeting of Ribosomes to the Rough Endoplasmic Reticulum in the Absence and Presence of the Nascent Polypeptide-associated Complex

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.

IκB Is a Substrate for a Selective Pathway of Lysosomal Proteolysis

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor κ B (IκB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IκB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IκB is directly transported into isolated lysosomes in a process that requires binding of IκB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IκB uptake by lysosomes. Ubiquitination and phosphorylation of IκB are not required for its targeting to lysosomes. The lysosomal degradation of IκB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IκB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IκB degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of IκB is increased. There are both short- and long-lived pools of IκB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IκB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IκB degradation in lysosomes. This selective pathway of lysosomal degradation of IκB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor κ B. In addition, the response of nuclear factor κ B to several stimuli increases when this lysosomal pathway of proteolysis is activated.

Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold, P < 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1+lin− adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11, flt3-ligand, and Steel factor. Moreover, the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures, long-term culture-initiating cells increased 7- ± 2-fold, myeloid colony-forming cells increased 140- ± 36-fold, and total nucleated cells increased 230- ± 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1+lin− marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension, suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119− CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult.

Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration

We report the construction of two novel Escherichia coli strains (DH1lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter. Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse. However, when the strains are transformed with a multicopy plasmid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolated and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are required on the plasmid, and antibiotics are not necessary for plasmid selection, making these strains a valuable tool for therapeutic DNA and recombinant protein production. We describe the construction of these strains and demonstrate plasmid selection and maintenance by repressor titration, using the new pORT plasmid vectors designed to facilitate recombinant DNA exploitation.

Human-caused environmental change: Impacts on plant diversity and evolution

Human-caused environmental changes are creating regional combinations of environmental conditions that, within the next 50 to 100 years, may fall outside the envelope within which many of the terrestrial plants of a region evolved. These environmental modifications might become a greater cause of global species extinction than direct habitat destruction. The environmental constraints undergoing human modification include levels of soil nitrogen, phosphorus, calcium and pH, atmospheric CO2, herbivore, pathogen, and predator densities, disturbance regimes, and climate. Extinction would occur because the physiologies, morphologies, and life histories of plants limit each species to being a superior competitor for a particular combination of environmental constraints. Changes in these constraints would favor a few species that would competitively displace many other species from a region. In the long-term, the “weedy” taxa that became the dominants of the novel conditions imposed by global change should become the progenitors of a series of new species that are progressively less weedy and better adapted to the new conditions. The relative importance of evolutionary versus community ecology responses to global environmental change would depend on the extent of regional and local recruitment limitation, and on whether the suite of human-imposed constraints were novel just regionally or on continental or global scales.

Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR

Sterol regulatory element-binding protein-1c (SREBP-1c) enhances transcription of genes encoding enzymes of unsaturated fatty acid biosynthesis in liver. SREBP-1c mRNA is known to increase when cells are treated with agonists of liver X receptor (LXR), a nuclear hormone receptor, and to decrease when cells are treated with unsaturated fatty acids, the end products of SREBP-1c action. Here we show that unsaturated fatty acids lower SREBP-1c mRNA levels in part by antagonizing the actions of LXR. In cultured rat hepatoma cells, arachidonic acid and other fatty acids competitively inhibited activation of the endogenous SREBP-1c gene by an LXR ligand. Arachidonate also blocked the activation of a synthetic LXR-dependent promoter in transfected human embryonic kidney-293 cells. In vitro, arachidonate and other unsaturated fatty acids competitively blocked activation of LXR, as reflected by a fluorescence polarization assay that measures ligand-dependent binding of LXR to a peptide derived from a coactivator. These data offer a potential mechanism that partially explains the long-known ability of dietary unsaturated fatty acids to decrease the synthesis and secretion of fatty acids and triglycerides in livers of humans and other animals.

Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomyces cerevisiae

Accumulation of misfolded proteins in the cell at high temperature may cause entry into a nonproliferating, heat-shocked state. The imino acid analog azetidine 2-carboxylic acid (AZC) is incorporated into cellular protein competitively with proline and can misfold proteins into which it is incorporated. AZC addition to budding yeast cells at concentrations sufficient to inhibit proliferation selectively activates heat shock factor (HSF). We find that AZC treatment fails to cause accumulation of glycogen and trehalose (Msn2/4-dependent processes) or to induce thermotolerance (a protein kinase C-dependent process). However, AZC-arrested cells can accumulate glycogen and trehalose and can acquire thermotolerance in response to a subsequent heat shock. We find that AZC treatment arrests cells in a viable state and that this arrest is reversible. We find that cells at high temperature or cells deficient in the ubiquitin-conjugating enzymes Ubc4 and Ubc5 are hypersensitive to AZC-induced proliferation arrest. We find that AZC treatment mimics temperature up-shift in arresting cells in G1 and represses expression of CLN1 and CLN2. Mutants with reduced G1 cyclin-Cdc28 activity are hypersensitive to AZC-induced proliferation arrest. Expression of the hyperstable Cln3–2 protein prevents G1 arrest upon AZC treatment and temperature up-shift. Finally, we find that the EXA3–1 mutation, encoding a defective HSF, prevents efficient G1 arrest in response to both temperature up-shift and AZC treatment. We conclude that nontoxic levels of misfolded proteins (induced by AZC treatment or by high temperature) selectively activate HSF, which is required for subsequent G1 arrest.

Herbicide Safener-Binding Protein of Maize1 : Purification, Cloning, and Expression of an Encoding cDNA

Dichloroacetamide safeners protect maize (Zea mays L.) against injury from chloroacetanilide and thiocarbamate herbicides. Etiolated maize seedlings have a high-affinity cytosolic-binding site for the safener [3H](R,S)-3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazol-idine ([3H]Saf), and this safener-binding activity (SafBA) is competitively inhibited by the herbicides. The safener-binding protein (SafBP), purified to homogeneity, has a relative molecular weight of 39,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 5.5. Antiserum raised against purified SafBP specifically recognizes a 39-kD protein in etiolated maize and sorghum (Sorghum bicolor L.), which have SafBA, but not in etiolated wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), or Arabidopsis, which lack SafBA. SafBP is most abundant in the coleoptile and scarcest in the leaves, consistent with the distribution of SafBA. SBP1, a cDNA encoding SafBP, was cloned using polymerase chain reaction primers based on purified proteolytic peptides. Extracts of Escherichia coli cells expressing SBP1 have strong [3H]Saf binding, which, like binding to the native maize protein, is competitively inhibited by the safener dichlormid and the herbicides S-ethyl dipropylthiocarbamate, alachlor, and metolachlor. SBP1 is predicted to encode a phenolic O-methyltransferase, but SafBP does not O-methylate catechol or caffeic acid. The acquisition of its encoding gene opens experimental approaches for the evaluation of the role of SafBP in response to the relevant safeners and herbicides.