DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways

DNA double-strand breaks (DSBs) in eukaryotic cells can be repaired by non-homologous end-joining or homologous recombination. The complex containing the Mre11, Rad50 and Nbs1 proteins has been implicated in both DSB repair pathways, even though they are mechanistically different. To get a better understanding of the properties of the human Mre11 (hMre11) protein, we investigated some of its biochemical activities. We found that hMre11 binds both double- and single-stranded (ss)DNA, with a preference for ssDNA. hMre11 does not require DNA ends for efficient binding. Interestingly, hMre11 mediates the annealing of complementary ssDNA molecules. In contrast to the annealing activity of the homologous recombination protein hRad52, the activity of hMre11 is abrogated by the ssDNA binding protein hRPA. We discuss the possible implications of the results for the role(s) of hMre11 in both DSB repair pathways.

Estimating the Excess Investment in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Leaves of Spring Wheat Grown under Elevated CO21

Wheat (Triticum aestivum L.) was grown under CO2 partial pressures of 36 and 70 Pa with two N-application regimes. Responses of photosynthesis to varying CO2 partial pressure were fitted to estimate the maximal carboxylation rate and the nonphotorespiratory respiration rate in flag and preceding leaves. The maximal carboxylation rate was proportional to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content, and the light-saturated photosynthetic rate at 70 Pa CO2 was proportional to the thylakoid ATP-synthase content. Potential photosynthetic rates at 70 Pa CO2 were calculated and compared with the observed values to estimate excess investment in Rubisco. The excess was greater in leaves grown with high N application than in those grown with low N application and declined as the leaves senesced. The fraction of Rubisco that was estimated to be in excess was strongly dependent on leaf N content, increasing from approximately 5% in leaves with 1 g N m−2 to approximately 40% in leaves with 2 g N m−2. Growth at elevated CO2 usually decreased the excess somewhat but only as a consequence of a general reduction in leaf N, since relationships between the amount of components and N content were unaffected by CO2. We conclude that there is scope for improving the N-use efficiency of C3 crop species under elevated CO2 conditions.

Visualization of Cavitated Vessels in Winter and Refilled Vessels in Spring in Diffuse-Porous Trees by Cryo-Scanning Electron Microscopy1

Xylem cavitation in winter and recovery from cavitation in the spring were visualized in two species of diffuse-porous trees, Betula platyphylla var. japonica Hara and Salix sachalinensis Fr. Schm., by cryo-scanning electron microscopy after freeze-fixation of living twigs. Water in the vessel lumina of the outer three annual rings of twigs of B. platyphylla var. japonica and of S. sachalinensis gradually disappeared during the period from January to March, an indication that cavitation occurs gradually in these species during the winter. In April, when no leaves had yet expanded, the lumina of most of the vessels of both species were filled with water. Many vessel lumina in twigs of both species were filled with water during the period from the subsequent growth season to the beginning of the next winter. These observations indicate that recovery in spring occurs before the onset of transpiration and that water transport through twigs occurs during the subsequent growing season. We found, moreover, that vessels repeat an annual cycle of winter cavitation and spring recovery from cavitation for several years until irreversible cavitation occurs.

Break-induced replication: A review and an example in budding yeast

Break-induced replication (BIR) is a nonreciprocal recombination-dependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5′ end of the LEU2 gene at one end was transformed into a strain in which the 5′ end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2+. Ade1+ Leu2+ transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADE2 added to the ends of a URA3 gene. Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADE2-adjacent sequences, including a nearby ARS, copied from chromosomal DNA. Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ.

The tight linkage between DNA replication and double-strand break repair in bacteriophage T4

Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.

Chromosomal double-strand break repair in Ku80-deficient cells.

The x-ray sensitive hamster cell line xrs-6 is deficient in DNA double-strand break (DSB) repair and exhibits impaired V(D)J recombination. The molecular defect in this line is in the 80-kDa subunit of the Ku autoantigen, a protein that binds to DNA ends and recruits the DNA-dependent protein kinase to DNA. Using an I-SceI endonuclease expression system, chromosomal DSB repair was examined in xrs-6 and parental CHO-K1 cell lines. A DSB in chromosomal DNA increased the yield of recombinants several thousand-fold above background in both the xrs-6 and CHO-K1 cells, with recombinational repair of DSBs occurring in as many as 1 of 100 cells electroporated with the endonuclease expression vector. Thus, recombinational repair of chromosomal DSBs can occur at substantial levels in mammalian cells and it is not grossly affected in our assay by a deficiency of the Ku autoantigen. Rejoining of broken chromosome ends (end-joining) near the site of the DSB was also examined. In contrast to recombinational repair, end-joining was found to be severely impaired in the xrs-6 cells. Thus, the Ku protein appears to play a critical role in only one of the chromosomal DSB repair pathways.

Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication.

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.

Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins.

The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression of RAD51 or RAD52. Virtually complete suppression is provided by the simultaneous overexpression of RAD51 and RAD52. This suppression occurs at 23 degrees C, where these mutants are more sensitive to x-rays, as well as at 30 degrees C and 36 degrees C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad51 and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks.

Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

The Vernacular Discourse of the "Arab Spring:" An Analysis of the Visual, the Embodied, and the Textual Rhetorics of the Karama Revolution

The Vernacular Discourse of the "Arab Spring" is a project that bridges the divide between the East and the West by offering new readings to Arab subjectivities. Through an analysis of the "Arab Spring" through the lens of vernacular discourse, it challenges the Euro-Americo-centric legacies of Orientalism in Western academia and the new wave of extremism in the Arab world by offering alternative representations of Arab bodies and subjectivities. To offer this new reading of the "Arab Spring," it explores the foundations of critical rhetoric as a theory and a practice and argues for a turn towards a critical vernacular discourse. The turn towards critical vernacular discourse is important as it urges the analyses of different artifacts produced by marginalized groups in order to understand their perspectives that have largely been foreclosed in traditional cultural studies research. Building on embodied/performative critical rhetoric, the vernacular discourses of the Arab revolutionary body examines other forms of knowledge productions that are not merely textual; more specifically, through data gathered in the Lhbib Bourguiba, Tunisia. This analysis of the political revolutionary body unveils the complexity underlining the discussion around issues of identity, agency and representation in the Middle East and North Africa, and calls for a critical study towards these issues in the region beyond the binary approach that has been practiced and applied by academics and media analysts. Hence, by analyzing vernacular discourse, this research locates a method of examining and theorizing the dialectic between agency, citizenry, and subjectivity through the study of how power structure is recreated and challenged through the use of the vernacular in revolutionary movements, as well as how marginalized groups construct their own subjectivities through the use of vernacular discourse. Therefore, highlighting the political prominence of evaluating the Arab Spring as a vernacular discourse is important in creating new ways of understanding communication in postcolonial/neocolonial settings.

Sovereignty and Intervention in the Middle East: From the Fall of the Ottoman Empire to the Arab Spring

This research provides an institutional explanation of the practices of external intervention in the Arab state system from the fall of the Ottoman Empire in 1922 to the Arab Spring. My explanation consists of two institutional variables: sovereignty and inter-state borders. I examine the changes in regional and international norms of sovereignty and their impact on the practices of external intervention in the Arab state system. I also examine the impact of the level of institutionalization of inter-state borders in the Arab World on the practices of external intervention. I argue that changes in regional and international norms of sovereignty and changes in the level of institutionalization of inter-state borders have constituted the significant variation over time in both the frequency and type of external intervention in the Arab state system from 1922 to the present. My institutional explanation and findings seriously challenge the traditional accounts of sovereignty and intervention in the Arab World, including the cultural perspectives that emphasize the conflict between sovereignty, Arabism, and Islam, the constructivist accounts that emphasize the regional norm of pan-Arabism, the comparative politics explanations that focus on the domestic material power of the Arab state, the post-colonial perspectives that emphasize the artificiality of the Arab state, and the realist accounts that focus on great powers and the regional distribution of power in the Middle East. This research also contributes to International Relations Theory. I construct a new analytical framework to study the relations between sovereignty, borders, and intervention, combining theoretical elements from the fields of Role Theory, Social Constructivism, and Institutionalization. Methodologically, this research includes both quantitative and qualitative analysis. I conduct content analysis of official documents of Arab states and the Arab League, Arabic press documents, and Arab political thought. I also utilize quantitative data sets on international intervention.

A Cross-Cultural Examination of the Use of Multimodal Metaphor in English and Arabic Cartoons of the ‘Arab Spring’ in Egypt

El estudio de Lakoff y Johnson Metáforas de la vida cotidiana (1980) ha dotado al campo de la metáfora de un renovado interés, y a éste siguieron numerosas publicaciones abordándolo desde diversas perspectivas. Lakoff y Johnson (1980) aseguran que la metáfora es, de hecho, una característica del pensamiento y, por consiguiente, ya no es una propiedad exclusiva del lenguaje. La metáfora sería en realidad un reflejo de cómo concebimos e interpretamos el mundo en que vivimos, en la medida en que está conformada por nuestras experiencias corporales, un fenómeno al que Lakoff y Johnson se refieren como la “mente corpórea”. La metáfora cumple la función de “comprender” y facilitar la interpretación de un ámbito o experiencia en los términos de otro ámbito o experiencia (Lakoff & Johnson, 1980: 36). Los conceptos abstractos son difíciles de ser pensados per se; consecuentemente, las personas recurren a la metáfora para concebirlos en los términos de otras nociones más concretas que están, en cierto grado, vinculadas a nuestras experiencias somáticas. Si bien no escasean las investigaciones en el campo de las metáforas, parece haber, sin embargo, cierto vacío en lo tocante a la manifestación de la metáfora en modos distintos del modo verbal. Forceville (2009: 19) afirma que uno de los principios fundamentales de la Teoría de la Metáfora Conceptual es la idea de que el variado empleo de metáforas por parte del ser humano sugiere “que piensa en gran medida metafóricamente”. Esto lleva a la conclusión de que la manifestación de la metáfora debería estar presente en los diversos modos del pensamiento, y no únicamente en el verbal. Estos otros modos de pensamiento incluyen, entre otros, las imágenes, la música, los sonidos y los gestos. Asimismo, la mayoría de estudios que se han realizado en el área de las metáforas multimodales ha sido aplicada al terreno de la publicidad. Los anunciantes parecen advertir el ! 2! poder que se invierte en las metáforas, y las usan profusamente para transmitir mensajes a los consumidores. No obstante, recientemente existe un nuevo interés por investigar el uso de metáforas multimodales en las caricaturas (véase, por ejemplo, El Refaie 2003, 2009; Schilperoord y Maes 2009; Yus 2009; Bergen 2003; Marin Aresse 2008). Las tiras cómicas se distinguen de los anuncios principalmente por el hecho de que las caricaturas transmiten una postura negativa frente a un sujeto particular, mientras que los anuncios presentan una actitud positiva. Igualmente, les diferencia el hecho de que las tiras cómicas requieren un conocimiento social y político específico. El presente escrito examina y compara el uso de metáforas multimodales en caricaturas políticas – tanto inglesas como árabes – que retratan la Primavera Árabe en Egipto, con el fin de determinar: (1) si la mayoría de caricaturas son conceptualmente específicas o culturalmente específicas; (2) los principales dominios fuente empleados por los caricaturistas ingleses y árabes para interpretar la Primavera Árabe; (3) si hay semejanzas o diferencias entre las caricaturas inglesas y las árabes en su elección del dominio meta; y (4) observar también cómo los distintos modos (aquí esencialmente los modos verbal y pictórico) contribuyen a representar la Primavera Árabe. El corpus para este estudio se compone de un total de 50 tiras cómicas, 25 de ellas inglesas y las 25 restantes árabes. En cada una de estas tiras debe haber al menos una metáfora que representa la Primavera Árabe o alguno de sus subtemas. Las tiras han sido seleccionadas aleatoriamente a través de Internet. Para el análisis, la investigadora ha seguido el marco teórico propuesto por Bounegru y Forceville (2011) en cuanto a la determinación de metáforas en general, así como la determinación de metáforas multimodales de tipo verbovisual en particular. Por cada tira cómica se ha llevado a cabo un análisis independiente, determinando el dominio fuente y el dominio meta, las metáforas, las asignaciones, qué se está representando verbalmente y qué se representa pictóricamente. Además, la autora ha analizado ! 3! posteriormente si estas metáforas son culturalmente específicas o no, y/o si están relacionadas con metáforas más genéricas o universales. El análisis que comprende este trabajo se divide en dos secciones. La primera parte es un estudio detallado de los dos corpora, abordando los distintos escenarios de los dominios fuente empleados por los dibujantes según su similitud. Esta sección presenta a su vez un análisis de los diversos modos que se utilizan para revelar el dominio fuente así como el dominio meta. Del mismo modo, se incluyen las metáforas específicas manejadas en cada tira cómica y, cuando se da el caso, aquellas metáforas genéricas o universales a las que remite. La segunda sección del análisis presenta una comparativa entre ambos corpora basada en el análisis expuesto en la primera parte. Además, trata varios de los fenómenos lingüísticos a los que han recurrido frecuentemente los dibujantes ingleses y árabes. Estos fenómenos son principalmente la metonimia y la personificación. Igualmente, en esta sección la autora investiga en mayor profundidad las metáforas usadas por los dibujantes ingleses y los árabes, determinando si son ora conceptualmente específicas, ora culturalmente específicas, a partir del hecho de que las metáforas sirven como herramienta para reconocer la forma en que personas distintas, o bien culturas distintas, interpretan varias cuestiones. Por ejemplo, a la hora de conceptualizar el futuro, algunas culturas lo representan espacialmente como delante del hablante, mientras que otras se refieren al mismo como localizado detrás del sujeto (Lakoff y Johnson 1980: 14). Este trabajo ha permitido varios hallazgos. En cuanto a las metáforas empleadas para representar la Primavera Árabe, tanto las tiras inglesas como las árabes han recurrido a una cierta variedad de metáforas. Algunas de las tiras presentan más de una metáfora operando a la vez. Los dibujantes ingleses y árabes parecen haberse apoyado siempre en el modo pictórico para presentar el dominio fuente, así como en el modo verbal para mostrar el dominio meta. ! 4! Además, respecto a la naturaleza de las metáforas que figuran en sendos corpora, casi todas las metáforas son conceptuales en cuanto que se corresponden con nuestras experiencias corporales; no hay, por otro lado, ninguna metáfora culturalmente específica. Asimismo, la única diferencia a este respecto entre ambos corpora es una variación en lo que constituye el prototipo de una categoría particular – aquí concretamente la categoría “primavera” en cada una de las culturas –. En las tiras inglesas, una flor es empleada para representar la primavera, mientras que en las árabes el elemento natural más frecuente para simbolizar la primavera es un árbol.

Effects of Annual Spring Flow Events on Abundance of Rio Grande Sillvery Minnow, Red Shiner, and Fathead Minnow with Notes on Recovery Needs for the Rio Grande Silvery Minnow

I investigated effects of annual spring flows on the abundance of Rio Grande silvery minnow, red shiner, and fathead minnow within two segments of the Middle Rio Grande (i.e., above and below the Rio Puerco) using 12 years of data. Rio Grande silvery minnow abundance was positively related to high spring flows and negatively related to low spring flows. Conversely, the abundance of the red shiner and fathead minnow were negatively related to high spring flows and positively related to low spring flows. The strength of these relationships varied with species and location but results from analyses below the Rio Puerco showed a stronger positive and negative relationship (with high and low spring flows, respectively) for Rio Grande silvery minnow.