Modelo de análisis organizativo para la mejora del desempeño de empresas sociales

La empresa social es un modelo organizativo que presenta un interesante potencial para resolver problemáticas sociales. La empresa social ha despertado interés tanto en países industrializados como en economías en vías de desarrollo porque representa un modelo dentro del capitalismo que persigue objetivos sociales mediante la realización de actividades de mercado (compra y venta de productos y/o servicios principalmente). A pesar de sus raíces lejanas en el tiempo se trata de un campo de conocimiento relativamente joven, donde la literatura académica presenta escasez de estudios empíricos. El desarrollo teórico para buscar claridad conceptual ha sido el principal caballo de batalla de los últimos años, y por tanto, se ha prestado poca atención a generar evidencias sobre cómo funcionan las empresas sociales y sobre sus claves de su éxito. Se considera que la mejora en la comprensión de este modelo organizativo pasa por la construcción de herramientas para que académicos y practicantes mejoren su conocimiento sobre los mecanismos internos de las empresas sociales. En este contexto nace la presente tesis doctoral sobre empresa social, que tiene por objetivo la creación de un marco de análisis que permita el estudio de las empresas sociales desde una dimensión organizativa, es decir, que aborde los elementos clave que describen el funcionamiento de este tipo de organizaciones. Para ello, en este trabajo se aborda la construcción del modelo para el análisis organizativo de las empresas sociales a partir del análisis semántico de las 45 principales definiciones de empresa social. A partir de este análisis se identifican dos dimensiones de análisis de la empresa social: -Cuatro principios, comunes a todas las manifestaciones del fenómeno, que recogen la esencia del concepto. -Ocho elementos organizativos específicos de la empresa social que describen la forma en la que cada iniciativa se implementa en un contexto determinado. Es decir, elementos de diseño presentes en diferente medida que dan lugar a tipologías de empresa social diferentes. Estos elementos son: la proposición de valor social, la búsqueda de impacto a largo plazo, la cultura organizativa, la conexión con los beneficiarios, el liderazgo emprendedor y los mecanismos de gobernanza, el ecosistema colaborativo, la estrategia empresarial y la orientación a la autosuficiencia económica. A partir de este marco de análisis, se construyen dos herramientas de diagnóstico que permiten su aplicación al estudio de empresas sociales: una tabla de indicadores para el análisis externo (por parte de un investigador ajeno a la organización) y un cuestionario de diagnóstico para el análisis interno (a través del personal de la empresa social objeto de estudio). Las herramientas intentan dar respuesta a la necesidad de desarrollar constructos para el estudio empírico de las empresas sociales. Para analizar la utilidad del modelo y de las herramientas se llevaron a cabo tres estudios de caso: -La empresa social ACCIONA Microenergía Perú que proporciona energía eléctrica a comunidades rurales aisladas en la región peruana de Cajamarca. -La empresa social Integra-e que propone un mecanismo de inserción socio-laboral en Madrid para jóvenes en riesgo de exclusión a través de la formación en Tecnologías de la Información y la Comunicación (TIC). -Un conjunto de redes de telecentros pertenecientes a la red LAC de la fundación Telecentres.org que proporcionan acceso a servicios de información (Internet entre otros) en diferentes países de Latinoamérica. La aplicación de las herramientas mostró ser útil en los tres estudios de caso para obtener una relación de evidencias con las que analizar la proximidad de una organización al ideal de empresa social. El ejercicio de análisis también resultó interesante como ejercicio reflexivo para las entidades participantes. Los resultados del cuestionario fueron especialmente interesantes en los telecentros de la Fundación Telecentre.org ya que al ser un estudio multicaso se pudo realizar un rico análisis estadístico sobre el funcionamiento de los telecentros y su desempeño. El estudio permitió identificar relaciones interesantes entre los ocho elementos de diseño del modelo propuesto y el desempeño de la organización. En particular, se detectó que para todos los casos estudiados: -La dimensión económica es la componente del desempeño que mayor desafíos plantea. -La existencia de una alta correlación entre el desempeño y siete de los ocho elementos organizativos del modelo. -La importancia de la cultura organizativa como elemento que explica el desempeño global de la organización y la satisfacción de los empleados. El campo de la empresa social presenta importantes retos de futuro, como la claridad conceptual, el desarrollo de estudios empíricos y la medida de su impacto social. El conocimiento de las claves organizativas puede ayudar a diseñar empresas sociales más robustas o a que organizaciones con fines sociales que no se basan en mecanismos de mercado consideren la posibilidad de incorporar éstos en su estrategia. ABSTRACT Social enterprise is an organizational model with a strong potential to help solving social problems. Recently, interest for the model has risen in both industrialized and developing countries because it is organized to achieve altruistic or social goals through market activities (mainly sales of products and services). Despite its historic roots, it is a relatively young field of research, where academic literature has little empirical data to accompany the theoretical development of social enterprise. Conceptual clarification has been the main challenge during the recent years, and there has been little attention given to generate evidence on how social enterprises operate and their keys to success. Progress in empirical study involves the construction of tools for researchers, in order to increase understanding of the internal mechanisms of social enterprises. This thesis aims to create a conceptual framework to study social enterprises from an organizational point of view, by analyzing the key elements that explain the operation and organization of this organizational model. The framework for the organizational analysis of social enterprises was built supported by the semantic analysis of 45 main definitions of social enterprise. The framework is divided into two dimensions: -There are four principles which capture the essence of the social enterprise concept, and are present in the manifestations of cases. -There are eight design elements which help analyze the characteristics of each particular social enterprise initiative: the social value proposition, social impact orientation, organizational culture, links to beneficiaries, entrepreneurial leadership, collaborative ecosystem, entrepreneurial strategy and orientation to economic self-sufficiency. Two diagnostic tools were developed to apply the framework to case studies: a scoreboard of indicators (to be used by the researcher during external analysis of the organization) and a questionnaire (to be answered by the social enterprise staff). The dissertation undertakes the study of three case studies: -ACCIONA Microenergia Peru, a social enterprise that provides electricity to isolated rural communities in the Peruvian region of Cajamarca. -Integra-e, a social enterprise located in Madrid that promotes socioprofessional integration of young people through training in ICT. -A sample of telecenters of the LAC network that provide access to information services (such as Internet) in Latin America. Applying the tools proved to be useful in all three cases, because it helped to obtain evidence to compare the proximity of an organization to an ideal type of social enterprise. In all the cases studied, the economic sustainability proved to be the biggest challenge for the organizations. The application of the questionnaire to the telecenters was especially informative because it was a multicase study which provided a rich statistical analysis on the performance of call centers. The study identified unique relationships between the model elements and the organziation performance. A statistical analysis shows a high correlation between performance and seven organizational elements described in the model. The organizational culture seems to be an important factor in explaining the overall organizational performance and employee satisfaction. The field of social enterprise has significant future challenges -such as conceptual clarity, the development of empirical studies and social impact assessment. A deep understanding of key organizational aspects of social enterprises can help in the design of more robust organizations and to bring success to social-purpose organizations.

Ciudades intermedias latinoamericanas ante modelos urbanos externos

En su proceso de crecimiento, las ciudades de América Latina y el Caribe (ALC) han tenido una vinculación histórica con la dinámica de implementación de herramientas, metodologías y proyectos urbanos, gestados en otros contextos, especialmente desde Europa (UE) y Estados Unidos (EE.UU). Desde la época colonial hasta hoy, la mayor parte de las ciudades de ALC han experimentado diversos tipos de influencia urbana externa, que han dejado huellas tangibles. Esta influencia ha ido variando a través de los años, desde la implantación directa de un modelo urbano, propia de la época colonial; hasta la importación de modelos urbanos total o parcialmente, de manera autónoma y ajena al origen de este. Actualmente, en ALC se han generado diversas iniciativas para abordar las necesidades urbanas desde sus especificidades, pero los instrumentos y proyectos vanguardistas utilizados en países desarrollados, siguen teniendo mayor fuerza de atracción y diseminación que las iniciativas vecinas. Se observa que las ciudades intermedias, que crecen con mayor velocidad que las grandes ciudades, también participan activamente en este proceso, aunque con otras limitaciones y condicionantes, diferentes a las encontradas en las grandes ciudades de la Región. ¿Por qué se produce esta dinámica?, ¿quiénes participan?, ¿cuáles son sus procesos?, ¿responden a las necesidades del complejo y diverso contexto urbano latinoamericano? Las Ciudades Intermedias Latinoamericanas ante los Modelos Urbanos Externos, es una investigación que aborda la dinámica de implementación de modelos de desarrollo urbanos1 exógenos, en ciudades emergentes que, en su proceso de crecimiento, tienden a repetir patrones de las grandes ciudades, y que en sí mismas, representan una oportunidad ante los desequilibrios territoriales de ALC. Esta dinámica, ha sido abordada por investigadores de diversas disciplinas, cuyos puntos de vista en muchas ocasiones no coinciden, pero revelan que estamos frente a una discusión de larga data, entre las visiones modernistas y las visiones identitarias de la historia urbana de ALC. Por ello, el trabajo recoge la evolución de los procesos de toma de decisiones, desarrollados bajo un efecto cascada (Global, Internacional, Regional, Nacional, Subnacional, Local), donde actualmente el nivel Local asciende a espacios del nivel Regional (ALC) e Internacional; pasando de meros receptores de políticas generadas en un plano superior derivadas de las relaciones geopolíticas y geoeconómicas de escala mundial; a ser sujetos proactivos del desarrollo de sus territorios. Para observar la concatenación de este proceso macro-disciplinar-micro (grosso modo), se plantea una herramienta metodológica de triangulación, desde la que se pueda visualizar el contexto en el que se produce la dinámica y como éste la condiciona. Con dicha metodología se abordará ALC como caso de estudio general, haciendo una aproximación al detalle en dos casos particulares: Ciudad Guayana (República Bolivariana de Venezuela) y Santiago de los Caballeros (República Dominicana). Estos casos, sumados a las observaciones de actores vinculados a la dinámica de transferencia de modelos urbanos, coadyuvarán en este esfuerzo de aproximación, para definir con más claridad un proceso que se incrementa y complejiza, en medio de la denominada “era de las ciudades”. ABSTRACT In its growth process, the cities of Latin America and the Caribbean (LAC) have had a historical link with the dynamics of implementation tools and engendered in other contexts, especially from Europe (EU) and the US methodologies (USA). From colonial times until today, most LAC cities have experienced various types of external urban influence that have left tangible traces. This influence has varied over the years, since the introduction of direct, typical of the colonial era; to import all or part independently and outside the origin of this urban model. Currently, in LAC they have generated various initiatives to address urban needs from its specificities, but notes that the instruments used in cutting-edge projects and developed countries, are still more attractive force and spread to neighboring initiatives. It is observed that the intermediate cities, which grow faster than big cities are also actively involved in this process, although with different constraints and other limitations to the big cities of the region. Why this dynamic occurs?, Who ?, which involved processes are ?, respond to the needs of complex and diverse Latin American urban context? Intermediate Latin American cities to external urban models, is a research that addresses the dynamics of exogenous implementation of urban development models in emerging cities in their growth process, they tend to repeat patterns of large cities, and represent a shot at LAC regional imbalances. This dynamic has been addressed by researchers from various disciplines, whose views often do not match, but show that we are facing a long-standing debate between modernists and identity visions on urban history of ALC. Therefore, the work shows the evolution of the processes of decision making, developed under a (global, international, regional, national, subnational, local) cascade effect, which currently stands at the Local level Regional level spaces (ALC) and International, from mere recipients of policies generated on a higher plane derived from the geopolitical and geo-economic relations worldwide; to be proactive in the development of their territories subject. To observe this process concatenation macro-micro-discipline (roughly), a triangulation methodology tool, from which it can view the context in which the dynamic occurs and how it affects what arises. ALC will address her as if general study, making an approach to detail two particular cases: Ciudad Guayana (Bolivarian Republic of Venezuela) and Santiago de los Caballeros (Dominican Republic). These cases, together with the comments of stakeholders involved in the dynamics of transfer of urban models, will assist in this effort approach to define more clearly a process that increases and more complex, amid the so-called "era of the cities".

Lactone-ring-cleaving enzyme: Genetic analysis, novel RNA editing, and evolutionary implications

A lactonohydrolase from Fusarium oxysporum AKU 3702 is an enzyme catalyzing the hydrolysis of aldonate lactones to the corresponding aldonic acids. The amino acid sequences of the NH2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR. An approximate 1,000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. A novel type of RNA editing, in which lactonohydrolase mRNA included the insertion of guanosine and cytidine residues, was observed. The predicted amino acid sequence of the cloned lactonohydrolase cDNA showed significant similarity to those of the gluconolactonase from Zymomonas mobilis, and paraoxonases from human and rabbit, forming a unique superfamily consisting of C-O cleaving enzymes and P-O cleaving enzymes. Lactonohydrolase was expressed under the control of the lac promoter in Escherichia coli.

cAMP receptor protein–cAMP plays a crucial role in glucose–lactose diauxie by activating the major glucose transporter gene in Escherichia coli

The inhibition of β-galactosidase expression in a medium containing both glucose and lactose is a typical example of the glucose effect in Escherichia coli. We studied the glucose effect in the lacL8UV5 promoter mutant, which is independent of cAMP and cAMP receptor protein (CRP). A strong inhibition of β-galactosidase expression by glucose and a diauxic growth were observed when the lacL8UV5 cells were grown on a glucose–lactose medium. The addition of isopropyl β-d-thiogalactoside to the culture medium eliminated the glucose effect. Disruption of the crr gene or overproduction of LacY also eliminated the glucose effect. These results are fully consistent with our previous finding that the glucose effect in wild-type cells growing in a glucose–lactose medium is not due to the reduction of CRP–cAMP levels but is due to the inducer exclusion. We found that the glucose effect in the lacL8UV5 cells was no longer observed when either the crp or the cya gene was disrupted. Evidence suggested that CRP–cAMP may not enhance directly the lac repressor action in vivo. Northern blot analysis revealed that the mRNA for ptsG, a major glucose transporter gene, was markedly reduced in a Δcrp or Δcya background. The constitutive expression of the ptsG gene by the introduction of a multicopy plasmid restored the glucose effect in Δcya or Δcrp cells. We conclude that CRP–cAMP plays a crucial role in inducer exclusion, which is responsible for the glucose–lactose diauxie, by activating the expression of the ptsG gene.

Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase α subunit

Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine- and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the α subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP α subunit. The A-tract sequence was protected by wild-type RNAP but not by α-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the −35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP αCTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.

Altered recognition mutants of the response regulator PhoB: A new genetic strategy for studying protein–protein interactions

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a “patch” near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and Pi signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6Kγ attP “genome targeting” suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.

Multiple pathways for SOS-induced mutagenesis in Escherichia coli: An overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA

dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated λ phage infecting UV-preirradiated bacterial cells (termed λUTM for λ untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for λUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F′lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P.

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.

Stationary-phase mutation in the bacterial chromosome: Recombination protein and DNA polymerase IV dependence

Several microbial systems have been shown to yield advantageous mutations in slowly growing or nongrowing cultures. In one assay system, the stationary-phase mutation mechanism differs from growth-dependent mutation, demonstrating that the two are different processes. This system assays reversion of a lac frameshift allele on an F′ plasmid in Escherichia coli. The stationary-phase mutation mechanism at lac requires recombination proteins of the RecBCD double-strand-break repair system and the inducible error-prone DNA polymerase IV, and the mutations are mostly −1 deletions in small mononucleotide repeats. This mutation mechanism is proposed to occur by DNA polymerase errors made during replication primed by recombinational double-strand-break repair. It has been suggested that this mechanism is confined to the F plasmid. However, the cells that acquire the adaptive mutations show hypermutation of unrelated chromosomal genes, suggesting that chromosomal sites also might experience recombination protein-dependent stationary-phase mutation. Here we test directly whether the stationary-phase mutations in the bacterial chromosome also occur via a recombination protein- and pol IV-dependent mechanism. We describe an assay for chromosomal mutation in cells carrying the F′ lac. We show that the chromosomal mutation is recombination protein- and pol IV-dependent and also is associated with general hypermutation. The data indicate that, at least in these male cells, recombination protein-dependent stationary-phase mutation is a mechanism of general inducible genetic change capable of affecting genes in the bacterial chromosome.

The sigma subunit of Escherichia coli RNA polymerase senses promoter spacing.

The promoters recognized by sigma 70, the primary sigma of Escherichia coli, consist of two highly conserved hexamers located at -10 and -35 bp from the start point of transcription, separated by a preferred spacing of 17 bp. sigma factors have two distinct DNA binding domains that recognize the two hexamer sequences. However, the component of RNA polymerase recognizing the length of the spacing between hexamers has not been determined. Using an equilibrium DNA binding competition assay, we demonstrate that a polypeptide of sigma 70 carrying both DNA binding domains is very sensitive to promoter spacing, whereas a sigma 70 polypeptide with only one DNA binding domain is not. Furthermore, a mutant sigma, selected for increasing transcription of the minimal lac promoter (18-bp spacer), has an altered response to promoter spacing in vivo and in vitro. Our data support the idea that sigma makes simultaneous, productive contacts at both the -10 and the -35 regions of the promoter and discerns the spacing between these conserved regions.

Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase.

Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(alphal,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(alpha1,3) Gal structure is formed by the enzyme Galbeta1,4GlcNAc3-alpha-D-galactosyltransferase [alpha(1,3)GT; EC 2.4.1.51], which transfers a galactose molecule to terminal N-acetyllactosamine (N-lac) present on various glycoproteins and glycolipids. The N-lac structure might be utilized as an acceptor by other glycosyltransferases such as Galbeta1,4GlcNAc 6-alpha-D-sialyltransferase [alpha(2,6)ST], Galbeta1,4GlcNAc 3-alpha-D-Sialyltransferase [alpha(2,3)ST], or Galbeta 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.691, etc. In this report we describe the competition between alpha(1,2)FT and alpha(1,3)GT in cells in culture and the generation of transgenic mice and transgenic pigs that express alpha(1,2)Fr leading to synthesis of Fucalpha,2Galbeta- (H antigen) and a concomitant decrease in the level of Gal(alpha1,3)Gal. As predicted, this resulted in reduced binding of xenoreactive natural antibodies to endothelial cells of transgenic mice and protection from complement mediated lysis.

Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice.

Transgenic mice carrying a bovine alpha-lactalbumin (alpha-lac) specific ribozyme gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat were generated and cross-bred with animals that highly express a bovine alpha-lac transgene (0.4 mg of alpha-lac/ml(-1) of milk). The ribozyme contains the hammerhead catalytic domain, flanked by 12-nt sequences complementary to the 3' untranslated region of bovine alpha-lac transcript. High-level expression of the ribozyme gene was detected by Northern blot analysis in the mammary gland of 7-8 day lactating transgenic mice, from 3 of 12 lines analyzed. Heterozygous expression of the ribozyme resulted in a reduction in the levels of the target mRNA to 78, 58, and 50% of that observed in the nonribozyme transgenic littermate controls for three independent lines. The ribozyme-mediated reduction in the levels of the bovine protein paralleled that observed for the mRNA, and was positively correlated with the level of expression of the ribozyme. In nonribozyme expressing transgenic mice, the level of bovine alpha-lac mRNA and protein was not affected. The specificity of this activity is demonstrated by the absence of a reduction in the levels of the endogenous murine alpha-lac mRNA or protein. These results demonstrate the feasibility of ribozyme-mediated down-regulation of highly-expressed transcripts in transgenic animals.

Molecularly engineered resistance to California serogroup virus replication in mosquito cells and mosquitoes.

Introduction of genetic elements derived from a viral pathogen's genome may be used to reduce the vectorial capacity of mosquitoes for that virus. A double subgenomic Sindbis virus expression system was utilized to transcribe sequences of LaCrosse (LAC) virus small (S) or medium (M) segment RNA in sense or antisense orientation; wild-type Sindbis and LaCrosse viruses have single-stranded RNA genomes, the former being positive sense and the latter being negative sense. Recombinant viruses were generated and used to infect Aedes albopictus (C6/36) mosquito cells, which were challenged with wild-type LAC virus and then assayed for LAC virus replication. Several recombinant viruses containing portions of the LAC S segment were capable of inducing varying degrees of interference to the challenge virus. Cells infected with TE/3'2J/ANTI-S virus, expressing full-length negative-sense S RNA of LAC virus, yielded 3-6 log10TCID50 (tissue culture 50% infective dose) less LAC virus per ml than did cells infected with a double subgenomic sindbis virus containing no LAC insert. When C6/36 cells infected with TE/3'2J/ANTI-S were challenged with closely related heterologous bunyaviruses, a similar inhibitory effect was seen. Adult Ae. triseriatus mosquitoes infected with TE/3'2J/ANTI-S were also resistant to challenge by LAC virus. Organs that were productively infected by the double subgenomic Sindbis virus expressing the LAC anti-S sequences demonstrated little LAC virus or antigen. These studies indicate that expression of carefully selected antiviral sequences derived from the pathogen's genome may result in efficacious molecular viral interference in mosquito cells and, more importantly, in mosquitoes.

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

Human immunodeficiency virus type-1 Tat is an integral component of the activated transcription-elongation complex.

The human immunodeficiency virus type 1 transactivator protein, Tat, stimulates transcriptional elongation from the viral long terminal repeat. To test whether Tat associates directly with activated transcription complexes, we have used the lac repressor protein (LacR) to "trap" elongating RNA polymerases. The arrested transcription complexes were purified by binding biotinylated templates to streptaviridin-coated magnetic beads. Transcription complexes were released from the magnetic beads following cleavage of the templates with restriction enzymes and were immunoblotted with antibodies to Tat, LacR and RNA polymerase II. The Tat protein copurified with RNA polymerase bound to wild-type templates but did not copurify with transcription complexes prepared by using templates carrying mutations in the transactivation response element (TAR) RNA. We conclude that Tat and cellular cofactors become attached to the transcription complex during its transit through TAR.