Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

Journal ArticleHeteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells

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

High frequency targeting of genes to specific sites in the mammalian genome

Journal ArticleWe corrected a defective gene residing in the chromosome of a mammalian cell by injecting into the nucleus copies of the same gene carrying a different mutation. We determined how the number, the arrangement, and the chromosomal position of the integrated gene, as well as the number of injected molecules influence the gene-targeting frequency

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

De la prosa de las relaciones a la épica gremial: Juan de Castellanos, "Elegías de varones ilustres de Indias" (1589)

The Elegías de varones ilustres de Indias (1589) by Juan de Castellanos are closely related to the contemporary Economy of Mercedes: a system in which deserving individuals (beneméritos) in the Americas aspire to a merced (reward, privilege) by means of an autobiographical report. In the Elegías we observe the transformation of a catalogue of beneméritos of the Kingdom of New Granada into an epic poem. The source of authority of this text is not the individual but the collective of the veterans. Castellanos found the model for his role as a spokesman of the “nobility” in Fernán Pérez de Guzmán, author of the Generaciones y semblanzas (ca. 1460).Las Elegías de varones ilustres de Indias (1589) de Juan de Castellanos se relacionan íntimamente con la economía de mercedes de la época: un sistema en el cual los beneméritos en las Indias pretenden una gratificación mediante relaciones autobiográficas. En las Elegías observamos la transformación de un catálogo de beneméritos novogranadinos en una epopeya cuya fuente de autoridad no es el individuo, sino el colectivo de los veteranos. Castellanos encontró el modelo para su papel de portavoz de la “nobleza” en Fernán Pérez de Guzmán, autor de las Generaciones y semblanzas (ca. 1460)

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