Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

Journal ArticleWe examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination

Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian. cell nuclei

Journal ArticleWe have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus

A persistent object manager for HEP

We propose to perform research in the area of a Persistant Object Manager for HEP. Persistant Objects are those which continue to exist upon process termination and may be accessed by other processes. It is expected that any system based upon this research will work primarily but not necessarily exclusively in an Object Oriented environment. Target applications include follow on or replacement products for existing packages such as GEANT, HEPDB, FATMEN, BHBOOK, experiment specific code event storage. In this respect, it is expected that more functionality will be required than simple persistance. It will be one of the goals of the of the project to define this extra layer of functionality. Strong emphasis will be placed on the use of standards and/or existing solutions wherever possible

Los «parerga» del manuscrito autógrafo: el ejemplo de “Basta callar” de Calderón (BNE, Res. 19)

Las tachaduras y atajos del manuscrito, en parte autógrafo, Res. 19 de la Biblioteca Nacional de España de la comedia “Basta callar” de Pedro Calderón de la Barca pueden interpretarse como parerga, o sea, elementos del marco suplementario del texto que es la condición de la significación de la obra. Las partes suprimidas indican que el suplemento del tema principal de la obra, la necesidad por parte de los poderosos de sacrificar sus pasiones, es la perversión, tal como se entendía en la época: «la corrupción de las costumbres».Manuscript Res. 19 of the Biblioteca Nacional de España, a copy of Calderón’s court spectacle “Basta callar”, partially written by the author himself, paradigmatically illustrates the logic and working of Jacques Derrida’s notion of the parergon («frame»). In the manuscript, we observe the (graphic) intrusion of the frames into the text. These frames were used by stage directors and/or the authors to mark sections to be suppressed in a particular representation. They both contain and suppress elements which are dispensable because they seem not to contribute to our understanding of the central notions of the play: The high nobility’s renunciation of passion for the sake of state and power. A detailed analysis of the barred materials shows that perversion, in its 17th-century meaning of a corruption of the «good customs» (ethical, sexual, and religious), is the necessary supplement of the logic of power

Calderón y la ‘mente cómica’ de su tiempo: el ejemplo de “El astrólogo fingido”

El ejemplo de El astrólogo fingido de Calderón de la Barca demuestra que la reconstrucción propuesta por Anthony Close de la mentalidad cómica colectiva del tiempo de Cervantes puede servir como marco apropiado para entender la comicidad en el teatro de comedias contemporáneo. La teoría del filósofo francés Henri Bergson sobre la risa aclara otras facetas de lo cómico en el teatro. Sobre todo en los aspectos metateatrales de El astrólogo fingido, observamos la rigidez mecánica bergsoniana que caracteriza los papeles teatrales, produciendo en los espectadores un alivio cómico en las normas de conducta y papeles sociales que experimentan en su vida cotidiana.The example of El astrólogo fingido by Calderón de la Barca shows that Anthony Close’s reconstruction of the collective comic mentality of the age of Cervantes is an appropriate frame for understanding the workings of humor in contemporary theater. French philosopher Henri Bergson’s theory of laughter sheds light on other aspects of humor in the theatre. Particularly in the metatheatrical elements of El astrólogo fingido we see that Bergson’s concept of mechanical rigidness is characteristic of theatrical roles, producing, in the spectators, comic relief from rigid norms of conduct and social roles they experience in everyday life

Assimilative, Autonomous, or Synergistic Visions: How Mediation Programs in Florida Address the Dilemma of Court Connection

Over the past twenty-five years, the state of Florida has been recognized across the United States as a leader in the development of court-connected alternative dispute resolution programs. Mediation, in particular, has flourished across the state, with one hundred eleven programs in place in family, civil, community, and dependency sectors. Administrative support and oversight for court-connected mediation programs are provided by The Florida Dispute Resolution Center (DRC) - the administrative arm of the Florida Supreme Court - housed within the Office of the State Courts Administrator. In collaboration with the DRC, we designed and conducted a benchmarking study of seven selected mediation programs in the family, civil and community sectors. Our focus in this article is on certain insights that emerged at the macro level regarding the very nature of court-connected mediation, as we analyzed and tried to develop explanations for our empirical findings. Specifically, we will address the fundamental value-based dilemma of court-connected mediation programs, three different approaches to addressing this dilemma through which mediation programs and the courts have forged their relationship in Florida, and some implications of these insights for practice and policy

The Precursors and Products of Justice Climates: Group Leader Antecedents and Employee Attitudinal Consequences

Drawing on the organizational justice, organizational climate, leadership and personality, and social comparison theory literatures, we develop hypotheses about the effects of leader personality on the development of three types of justice climates (e.g., procedural, interpersonal, and informational), and the moderating effects of these climates on individual level justice- attitude relationships. Largely consistent with the theoretically-derived hypotheses, the results showed that leader (a) agreeableness was positively related to procedural, interpersonal and informational justice climates, (b) conscientiousness was positively related to a procedural justice climate, and (c) neuroticism was negatively related to all three types of justice climates. Further, consistent with social comparison theory, multilevel data analyses revealed that the relationship between individual justice perceptions and job attitudes (e.g., job satisfaction, commitment) was moderated by justice climate such that the relationships were stronger when justice climate was high

Organisational justice:new insights from behavioural ethics

Both organizational justice and behavioural ethics are concerned with questions of 'right and wrong' in the context of work organizations. Until recently they have developed largely independently of each other, choosing to focus on subtly different concerns, constructs and research questions. The last few years have, however, witnessed a significant growth in theoretical and empirical research integrating these closely related academic specialities. We review the organizational justice literature, illustrating the impact of behavioural ethics research on important fairness questions. We argue that organizational justice research is focused on four reoccurring issues: (i) why justice at work matters to individuals; (ii) how justice judgements are formed; (iii) the consequences of injustice; and (iv) the factors antecedent to justice perceptions. Current and future justice research has begun and will continue borrowing from the behavioural ethics literature in answering these questions

TASK HANDOFF BETWEEN HUMANS AND AUTOMATION

The Department of Defense (DOD) seeks to incorporate human-automation teaming to decrease human operators’ cognitive workload, especially in the context of future vertical lift (FVL). Researchers created a “wizard of oz” study to observe human behavior changes as task difficulty and levels of automation increased. The platform used for the study was a firefighting strategy software game called C3Fire. Participants were paired with a confederate acting as an automated agent to observe the participant’s behavior in a human-automation team. The independent variables were automation level (within; low, medium, high) and queuing (between; uncued, cued). The dependent variables were the number of messages transmitted to the confederate, the number of tasks embedded in those messages (tasks handed off), and the participant’s self-reported cognitive workload score. The study results indicated that as the confederate increased its scripted level of automation, the number of tasks handed off to automation increased. However, the number of messages transmitted to automation and the subjective cognitive workload remained the same. The study’s findings suggest that while human operators were able to bundle tasks, cognitive workload remained relatively unchanged. The results imply that the automation level may have less impact on cognitive workload than anticipated.Major, United States ArmyCaptain, United States ArmyCaptain, United States ArmyCaptain, United States ArmyCaptain, United States ArmyApproved for public release. Distribution is unlimited