Quality assurance systems in the production and marketing of fresh peaches.

Due to the fast rate of peach post-harvest ripening, damage due to mechanical handling, externally appreciated as bruises and soft areas, is a real problem that leads to an early harvesting and poor quality of the fruits, as perceived by the consumers. More and more, the European consumer asks for good taste and freshness of fruits and vegetables, and these quality factors are not included in standards, nor in most of the producers' practices. Fruit processing and marketing centres (co-operatives) are increasingly interested in adopting quality controls in their processes. ISO 9000 procedures are being applied in some food areas, primarily milk and meat processors, but no generalised procedures have been developed until the present time to be applied to fresh product processes. All different peach and nectarine varieties that are harvested and handled in Murcia cooperatives and sold in a large supermarket in Madrid were analysed during the whole 1997 season (early May to late August). A total number of 78 samples of 25 fruits (co-operative) or 10 fruits (market), were tested in the laboratory for mechanical, optical, chemical and tasting quality. The variability and relationships between all these quality parameters are presented and discussed, and sampling unit sizes which would be advisable for quality control are calculated.

Key quality factors and travellers profiles at interurban interchanges

La planificación y las políticas de transporte no pueden descuidar la calidad del servicio, considerando que influye notablemente en el cambio modal del coche hacia otros medios de transporte más sostenibles. El concepto se aplica también a los intercambiadores de transporte público, los nodos del sistema donde se cruzan las distintas redes del transporte público y privado. Aunque se han logrado numerosos avances para medir y evaluar la calidad en el sector del transporte público, se han dedicado relativamente pocos esfuerzos a investigar estos aspectos relacionados con la calidad de los intercambiadores del transporte público. Este trabajo de investigación se concentra en la calidad del servicio de la transferencia modal en los intercambiadores interurbanos, según la perspectiva de los viajeros. Su objetivo es identificar los factores clave de la calidad del servicio y los perfiles de los viajeros en los intercambiadores. La investigación es exploratoria y ofrece información acerca de la percepción de los viajeros intermodales relacionada con los aspectos de la calidad, aportando nuevos elementos y datos para adentrarse en estudios más detallados. La metodología del trabajo combina técnicas de análisis estadístico multivariante para analizar los datos de las encuestas sobre la satisfacción de los clientes y se subdivide en tres etapas. En primer lugar, se ha implementado el análisis de correspondencias múltiples para explorar los constructos latentes relacionados con la satisfacción de las características cualitativas de los intercambiadores interurbanos, identificando así los factores clave de la calidad. En segundo lugar, se ha aplicado un análisis de conglomerados de k-medias sobre los factores clave de calidad para clasificar a los viajeros en grupos de usuarios de transportes homogéneos, de acuerdo con su percepción de satisfacción, identificando de este modo los perfiles de los viajeros. Por último, se han formulado sugerencias y recomendaciones sobre la calidad para respaldar la formulación de políticas, estableciendo las prioridades para los intercambiadores interurbanos. La metodología se aplicó en cuatro intercambiadores interurbanos (estaciones de ferrocarriles o de autobuses ) en Madrid, Zaragoza, Gothenburg y Lion, analizando los datos recogidos mediante una encuesta de satisfacción del cliente llevada a cabo en 2011 en los cuatro casos de estudio, donde se interconectan distintos medios de transporte público y privado, de corta y larga distancia. Se recogieron datos sobre la satisfacción de los viajeros con 26 criterios de calidad, así como información sobre aspectos socio-económicos y pautas de comportamiento de viajes. Mediante el análisis de correspondencias múltiples se identificaron 4-5 factores clave de calidad en cada intercambiador, que se asocian principalmente con el sistema de emisión de billetes, el confort y la interconexión, mientras que los viajeros no perciben los temas clásicos como la información. Mediante el análisis de conglomerados se identificaron 2-5 perfiles de viajeros en cada intercambiador. Se reconocieron dos grupos de viajeros en casi todos los casos de estudio: viajeros de cercanía/trabajadores y turistas. Por lo que concierne a las prioridades para apoyar a las partes interesadas en la formulación de políticas, la expedición de billetes es el factor clave para los intercambiadores interurbanos españoles, mientras que la interconexión y los aspectos temporales se destacan en los intercambiadores de Francia y Suecia. Quality of Service can not be neglected in public transport planning and policy making, since it strongly influences modal shifts from car to more sustainable modes. This concept is also related to Public Transport interchanges, the nodes of the transport system where the different sub-systems of public passenger transport and personal vehicles meet. Although a lot of progress has been generally done to measure and assess quality in public transport sector, relatively little investigation has been conducted on quality at PT interchanges. This research work focusses on Quality of Service in the use of transfer facilities at interurban interchanges, according to current travellers’ perspective. It aims at identifying key quality factors and travellers profiles at interurban interchanges. The research is exploratory and offers insight into intermodal travellers’ perception on quality aspects, providing new elements and inputs for more definitive investigation. The methodology of the work combines multivariate statistical techniques to analyse data from customer satisfaction surveys and is subdivided in three steps. Firstly, multiple correspondence analysis was performed to explore latent constructs as concern satisfaction of quality attributes at interurban interchanges, thus identifying the so-called Key Quality Factor. Secondly, k-means cluster analysis was implemented on the key quality factors to classify travellers in homogeneous groups of transport users, according to their perception of satisfaction, thus identifying the so-called Travellers Profiles. Finally, hints and recommendations on quality were identified to support policy making, setting priorities for interurban interchanges. The methodology was applied at four interurban interchanges in Madrid, Zaragoza, Gothenburg and Lyon, analysing the data collected through a customer satisfaction survey carried out in 2011 at the four railway or bus stations where different modes of public and private transport are interconnected covering both short and long trips. Data on travellers’ satisfaction with 26 quality attributes were collected, as well as information on socio-economical and travel patterns. Through multiple correspondence analysis were identified 4-5 key quality factors per interchange. They are mainly related to ticketing, comfort and connectivity, while classical issues, as information, are not perceived as important by travellers’. Through cluster analysis were identified 2-5 travellers profiles per interchange. Two groups of travellers can be found in almost all case studies: commuter / business travellers and holiday travellers. As regards the priorities to support stakeholders in policy making, ticketing is the key-issue for the Spanish interurban interchanges, while connectivity and temporal issues emerge in the French and Swedish case studies.

Instrumental quality assessment of fresh peaches: Optical and mechanical parameters.

In developing instrumentation for the measurement of fruit quality, there is the need for fast and non-destructive devices, based on sensors, to be installed on-line. In the case of some fruits, like peaches, post-harvest ripeness, which is closely related to high quality for the consumer, is a priority. During ripening, external appearance (colour) and internal mechanical (firmness) and chemical (sugars and acids) quality are main features that evolve rapidly from and unripe to a ripe (high quality) stage. When considering the evolution of fruit quality in this scheme, external colour and firmness are shown to evolve in a parallel pattern, if monitored from the time of harvest to full consumer ripeness ( Rood, 1957; Crisosto et al, 1995; Kader, 1996). The visible (VIS) reflectance spectrum is a fast and easy reference that can be used to estimate quality of peaches, if we could show it to be reliably correlated with peach ripening rate during postharvest (Genard et al. 1994; Moras, 1995; Delwiche and Baumgartner, 1983; Delwiche et al. 1987; Slaughter, 1995; Lleo et al., 1998). Taste, described as an expert acceptance score, improves with ripeness (firmness and colour evolution), when considering the fruits on the tree, and also post-harvest.

Influence of origin of the beans on protein quality and nutritive value of commercial soybean meals.

Chemical composition and correlations between chemical analyses and protein quality of 454 batches of SBM of 3 different origins (USA, n=168; Brazil (BRA), n=139, and Argentine (ARG), n=147) were studied. Samples were collected during a 6-yr period. SBM from USA had more CP, sucrose and stachyose and less NDF (P<0.001) than SBM from ARG and BRA. CP content was negatively related (P<0.001) with sucrose for USA meals and with NDF for ARG and BRA meals. Also, P content was positively related (P<0.01) with CP content of the meals. PDI and KOH solubility were higher (P<0.001) for USA than for ARG or BRA SBM, values that were positively related (P<0.001) with trypsin inhibitor activity of the meals. In addition, USA meals had more lys, met+cys, thr, and trp than BRA and ARG meals (P < 0.001). Per unit of CP, lys content was negatively related (P<0.001) with CP content for USA, positively for BRA, and no relations was found for ARG meals. It is concluded that nutritive values and protein quality of the meals varied widely among soybean origins. Consequently, the origin of the beans should be considered in the evaluation of the nutritive value of commercial SBM for non-ruminant animals.

Deposition reactors for solar grade silicon: a comparative thermal analysis of a Siemens reactor and a fluidized bed reactor

Polysilicon production costs contribute approximately to 25-33% of the overall cost of the solar panels and a similar fraction of the total energy invested in their fabrication. Understanding the energy losses and the behaviour of process temperature is an essential requirement as one moves forward to design and build large scale polysilicon manufacturing plants. In this paper we present thermal models for two processes for poly production, viz., the Siemens process using trichlorosilane (TCS) as precursor and the fluid bed process using silane (monosilane, MS).We validate the models with some experimental measurements on prototype laboratory reactors relating the temperature profiles to product quality. A model sensitivity analysis is also performed, and the efects of some key parameters such as reactor wall emissivity, gas distributor temperature, etc., on temperature distribution and product quality are examined. The information presented in this paper is useful for further understanding of the strengths and weaknesses of both deposition technologies, and will help in optimal temperature profiling of these systems aiming at lowering production costs without compromising the solar cell quality.

Proposal of a trust and reputation model to provide recommendation in open quality oriented environments

El auge y penetración de las nuevas tecnologías junto con la llamada Web Social están cambiando la forma en la que accedemos a la medicina. Cada vez más pacientes y profesionales de la medicina están creando y consumiendo recursos digitales de contenido clínico a través de Internet, surgiendo el problema de cómo asegurar la fiabilidad de estos recursos. Además, un nuevo concepto está apareciendo, el de pervasive healthcare o sanidad ubicua, motivado por pacientes que demandan un acceso a los servicios sanitarios en todo momento y en todo lugar. Este nuevo escenario lleva aparejado un problema de confianza en los proveedores de servicios sanitarios. Las plataformas de eLearning se están erigiendo como paradigma de esta nueva Medicina 2.0 ya que proveen un servicio abierto a la vez que controlado/supervisado a recursos digitales, y facilitan las interacciones y consultas entre usuarios, suponiendo una buena aproximación para esta sanidad ubicua. En estos entornos los problemas de fiabilidad y confianza pueden ser solventados mediante la implementación de mecanismos de recomendación de recursos y personas de manera confiable. Tradicionalmente las plataformas de eLearning ya cuentan con mecanismos de recomendación, si bien están más enfocados a la recomendación de recursos. Para la recomendación de usuarios es necesario acudir a mecanismos más elaborados como son los sistemas de confianza y reputación (trust and reputation) En ambos casos, tanto la recomendación de recursos como el cálculo de la reputación de los usuarios se realiza teniendo en cuenta criterios principalmente subjetivos como son las opiniones de los usuarios. En esta tesis doctoral proponemos un nuevo modelo de confianza y reputación que combina evaluaciones automáticas de los recursos digitales en una plataforma de eLearning, con las opiniones vertidas por los usuarios como resultado de las interacciones con otros usuarios o después de consumir un recurso. El enfoque seguido presenta la novedad de la combinación de una parte objetiva con otra subjetiva, persiguiendo mitigar el efecto de posibles castigos subjetivos por parte de usuarios malintencionados, a la vez que enriquecer las evaluaciones objetivas con información adicional acerca de la capacidad pedagógica del recurso o de la persona. El resultado son recomendaciones siempre adaptadas a los requisitos de los usuarios, y de la máxima calidad tanto técnica como educativa. Esta nueva aproximación requiere una nueva herramienta para su validación in-silico, al no existir ninguna aplicación que permita la simulación de plataformas de eLearning con mecanismos de recomendación de recursos y personas, donde además los recursos sean evaluados objetivamente. Este trabajo de investigación propone pues una nueva herramienta, basada en el paradigma de programación orientada a agentes inteligentes para el modelado de comportamientos complejos de usuarios en plataformas de eLearning. Además, la herramienta permite también la simulación del funcionamiento de este tipo de entornos dedicados al intercambio de conocimiento. La evaluación del trabajo propuesto en este documento de tesis se ha realizado de manera iterativa a lo largo de diferentes escenarios en los que se ha situado al sistema frente a una amplia gama de comportamientos de usuarios. Se ha comparado el rendimiento del modelo de confianza y reputación propuesto frente a dos modos de recomendación tradicionales: a) utilizando sólo las opiniones subjetivas de los usuarios para el cálculo de la reputación y por extensión la recomendación; y b) teniendo en cuenta sólo la calidad objetiva del recurso sin hacer ningún cálculo de reputación. Los resultados obtenidos nos permiten afirmar que el modelo desarrollado mejora la recomendación ofrecida por las aproximaciones tradicionales, mostrando una mayor flexibilidad y capacidad de adaptación a diferentes situaciones. Además, el modelo propuesto es capaz de asegurar la recomendación de nuevos usuarios entrando al sistema frente a la nula recomendación para estos usuarios presentada por el modo de recomendación predominante en otras plataformas que basan la recomendación sólo en las opiniones de otros usuarios. Por último, el paradigma de agentes inteligentes ha probado su valía a la hora de modelar plataformas virtuales complejas orientadas al intercambio de conocimiento, especialmente a la hora de modelar y simular el comportamiento de los usuarios de estos entornos. La herramienta de simulación desarrollada ha permitido la evaluación del modelo de confianza y reputación propuesto en esta tesis en una amplia gama de situaciones diferentes. ABSTRACT Internet is changing everything, and this revolution is especially present in traditionally offline spaces such as medicine. In recent years health consumers and health service providers are actively creating and consuming Web contents stimulated by the emergence of the Social Web. Reliability stands out as the main concern when accessing the overwhelming amount of information available online. Along with this new way of accessing the medicine, new concepts like ubiquitous or pervasive healthcare are appearing. Trustworthiness assessment is gaining relevance: open health provisioning systems require mechanisms that help evaluating individuals’ reputation in pursuit of introducing safety to these open and dynamic environments. Technical Enhanced Learning (TEL) -commonly known as eLearning- platforms arise as a paradigm of this Medicine 2.0. They provide an open while controlled/supervised access to resources generated and shared by users, enhancing what it is being called informal learning. TEL systems also facilitate direct interactions amongst users for consultation, resulting in a good approach to ubiquitous healthcare. The aforementioned reliability and trustworthiness problems can be faced by the implementation of mechanisms for the trusted recommendation of both resources and healthcare services providers. Traditionally, eLearning platforms already integrate recommendation mechanisms, although this recommendations are basically focused on providing an ordered classifications of resources. For users’ recommendation, the implementation of trust and reputation systems appears as the best solution. Nevertheless, both approaches base the recommendation on the information from the subjective opinions of other users of the platform regarding the resources or the users. In this PhD work a novel approach is presented for the recommendation of both resources and users within open environments focused on knowledge exchange, as it is the case of TEL systems for ubiquitous healthcare. The proposed solution adds the objective evaluation of the resources to the traditional subjective personal opinions to estimate the reputation of the resources and of the users of the system. This combined measure, along with the reliability of that calculation, is used to provide trusted recommendations. The integration of opinions and evaluations, subjective and objective, allows the model to defend itself against misbehaviours. Furthermore, it also allows ‘colouring’ cold evaluation values by providing additional quality information such as the educational capacities of a digital resource in an eLearning system. As a result, the recommendations are always adapted to user requirements, and of the maximum technical and educational quality. To our knowledge, the combination of objective assessments and subjective opinions to provide recommendation has not been considered before in the literature. Therefore, for the evaluation of the trust and reputation model defined in this PhD thesis, a new simulation tool will be developed following the agent-oriented programming paradigm. The multi-agent approach allows an easy modelling of independent and proactive behaviours for the simulation of users of the system, conforming a faithful resemblance of real users of TEL platforms. For the evaluation of the proposed work, an iterative approach have been followed, testing the performance of the trust and reputation model while providing recommendation in a varied range of scenarios. A comparison with two traditional recommendation mechanisms was performed: a) using only users’ past opinions about a resource and/or other users; and b) not using any reputation assessment and providing the recommendation considering directly the objective quality of the resources. The results show that the developed model improves traditional approaches at providing recommendations in Technology Enhanced Learning (TEL) platforms, presenting a higher adaptability to different situations, whereas traditional approaches only have good results under favourable conditions. Furthermore the promotion period mechanism implemented successfully helps new users in the system to be recommended for direct interactions as well as the resources created by them. On the contrary OnlyOpinions fails completely and new users are never recommended, while traditional approaches only work partially. Finally, the agent-oriented programming (AOP) paradigm has proven its validity at modelling users’ behaviours in TEL platforms. Intelligent software agents’ characteristics matched the main requirements of the simulation tool. The proactivity, sociability and adaptability of the developed agents allowed reproducing real users’ actions and attitudes through the diverse situations defined in the evaluation framework. The result were independent users, accessing to different resources and communicating amongst them to fulfil their needs, basing these interactions on the recommendations provided by the reputation engine.

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

Versatile 5′ phosphoryl coupling of small and large molecules to an RNA

A Ca2+-requiring catalytic RNA is shown to create 5′ phosphate–phosphate linkages with all nucleotides and coenzymes including CoA, nicotinamide adenine dinucleotide phosphate, thiamine phosphate, thiamine pyrophosphate, and flavin mononucleotide. In addition to these small molecules, macromolecules such as RNAs with 5′-diphosphates, and nonnucleotide molecules like Nɛ-phosphate arginine and 6-phosphate gluconic acid also react. That is, the self-capping RNA isolate 6 is an apparently universal 5′ phosphate-linker, reacting with any nucleophile containing an unblocked phosphate. These RNA reactions demonstrate a unique RNA catalytic capability and imply versatile and specific posttranscriptional RNA modification by RNA catalysis.

Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal NeuronsV⃞

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA.

Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression in response to Tat is dependent on an element downstream of the HIV-1 transcriptional initiation site designated the trans-activating region (TAR). TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat activation. In the absence of Tat, the HIV-1 promoter generates so-called short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized. Tat effects on transcriptional elongation are likely due to alterations in the elongation properties of RNA polymerase II. In this study we demonstrated that a set of cellular cofactors that modulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymerase II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initiate in vitro transcription from the HIV-1 long terminal repeat. RNA gel retardation analysis with wild-type and mutant TAR RNAs indicated that the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II. The addition of wild-type but not mutant Tat protein to gel retardation analysis with TAR RNA and RNA polymerase II resulted in the loss of binding of RNA polymerase II binding to TAR RNA. These results suggest that Tat may function to alter RNA polymerase II, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.

Precursor of C4 antisense RNA of bacteriophages P1 and P7 is a substrate for RNase P of Escherichia coli.

The C4 repressor of the temperate bacteriophages P1 and P7 inhibits antirepressor (Ant) synthesis and is essential for establishment and maintenance of lysogeny. C4 is an antisense RNA acting on a target, Ant mRNA, which is transcribed from the same promoter. The antisense-target RNA interaction requires processing of C4 RNA from a precursor RNA. Here we show that 5' maturation of C4 RNA in vivo depends on RNase P. In vitro, Escherichia coli RNase P and its catalytic RNA subunit (M1 RNA) can generate the mature 5' end of C4 RNA from P1 by a single endonucleolytic cut, whereas RNase P from the E. coli rnpA49 mutant, carrying a missense mutation in the RNase P protein subunit, is defective in the 5' maturation of C4 RNA. Primer extension analysis of RNA transcribed in vivo from a plasmid carrying the P1 c4 gene revealed that 5'-mature C4 RNA was the predominant species in rnpA+ bacteria, whereas virtually no mature C4 RNA was found in the temperature-sensitive rnpA49 strain at the restrictive temperature. Instead, C4 RNA molecules carrying up to five extra nucleotides beyond the 5' end accumulated. The same phenotype was observed in rnpA+ bacteria which harbored a plasmid carrying a P7 c4 mutant gene with a single C-->G base substitution in the structural homologue to the CCA 3' end of tRNAs. Implications of C4 RNA processing for the lysis/lysogeny decision process of bacteriophages P1 and P7 are discussed.

Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

In this report we show that yeast expressing brome mosaic virus (BMV) replication proteins 1a and 2a and replicating a BMV RNA3 derivative can be extracted to yield a template-dependent BMV RNA-dependent RNA polymerase (RdRp) able to synthesize (-)-strand RNA from BMV (+)-strand RNA templates added in vitro. This virus-specific yeast-derived RdRp mirrored the template selectivity and other characteristics of RdRp from BMV-infected plants. Equivalent extracts from yeast expressing 1a and 2a but lacking RNA3 contained normal amounts of 1a and 2a but had no RdRp activity on BMV RNAs added in vitro. To determine which RNA3 sequences were required in vivo to yield RdRp activity, we tested deletions throughout RNA3, including the 5',3', and intercistronic noncoding regions, which contain the cis-acting elements required for RNA3 replication in vivo. RdRp activity was obtained only from cells expressing 1a, 2a, and RNA3 derivatives retaining both 3' and intercistronic noncoding sequences. Strong correlation between extracted RdRp activity and BMV (-)-strand RNA accumulation in vivo was found for all RNA3 derivatives tested. Thus, extractable in vitro RdRp activity paralleled formation of a complex capable of viral RNA synthesis in vivo. The results suggest that assembly of active RdRp requires not only viral proteins but also viral RNA, either to directly contribute some nontemplate function or to recruit essential host factors into the RdRp complex and that sequences at both the 3'-terminal initiation site and distant internal sites of RNA3 templates may participate in RdRp assembly and initiation of (-)-strand synthesis.

NusA interferes with interactions between the nascent RNA and the C-terminal domain of the alpha subunit of RNA polymerase in Escherichia coli transcription complexes.

The effects of NusA on the RNA polymerase contacts made by nucleotides at internal positions in the nascent RNA in Escherichia coli transcription complexes were analyzed by using the photocrosslinking nucleotide analog 5-[(4-azidophenacyl) thio]-UMP. It was placed at nucleotides between +6 and +15 in RNA transcribed from the phage lambda PR' promoter. Crosslinks of analog in these positions in RNAs which contained either 15, 28, 29, or 49 nt were examined. Contacts between the nascent RNA and proteins in the transcription complex were analyzed as the RNA was elongated, by placing the crosslinker nearest the 5' end of the RNA 10, 23, 24, or 44 nt away from the 3' end. The beta or beta' subunit of polymerase, and NusA when added, were contacted by RNA from 15 to 49 nt long. When the upstream crosslinker was 24 nt from the 3" end of the RNA (29-nt RNA), alpha was also contacted in the absence of NusA. The addition of NusA prevented RNA crosslinking to alpha. When the crosslinker was 44 nt from the 3' end (49-nt RNA), alpha crosslinks were still observed, but crosslinks to beta or beta' and NusA were greatly diminished. RNA crosslinking to alpha, and loss of this crosslink when NusA was added, was observed in the presence of NusB, NusE, and NusG and when transcription was carried out in the presence of an E. coli S100 cell extract. Peptide mapping localized the RNA interactions to the C-terminal domain of alpha.