Control mechanisms of bacteriophage Φ29 DNA expression

The phage Φ29 regulatory protein p4 activates the late promoter A3 by stabilizing the binding of Bacillus subtilis RNA polymerase (RNAP) as a closed complex. Interaction between the two proteins occurs through amino acid Arg120 in protein p4 and the C-terminal domain of the RNAP α subunit (α-CTD). In addition to its role as activator of the late transcription, protein p4 represses early transcription from the A2b and A2c promoters, that are divergently transcribed. Binding of p4 to its recognition site at the A3 promoter displaces the RNAP from promoter A2b, both by steric hindrance and by the curvature induced upon p4 binding. At the A2c promoter, the RNAP cooperates with p4 binding in such a way that promoter clearance is prevented. Interestingly, amino acid Arg120 in p4 and the α-CTD in B. subtilis RNAP are involved in the interactions that lead to transcription repression at promoter A2c. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. In the absence of a –35 consensus box for σA-RNAP activation was observed, while in its presence repression occurred. The results support the idea that overstabilization of RNAP at the promoter over a threshold level leads to repression

Desplazamiento y ciudadanía, un estudio de caso^

Este trabajo de grado indagó por las transformaciones de las relaciones del Estado y la sociedad civil a partir del impacto producido por el desplazamiento forzado y su incidencia en la construcción de ciudadanía en el caso de la población del corregimiento de Sabaneta (Magangué) que se ha desplazado hacia el municipio de Buenavista desde el año 2002. Se desarrolló en tres capítulos: el primero introduce al lector en los orígenes de Sabaneta y llega hasta la decadencia de la ANUC. También describe las relaciones entre el Estado y la sociedad civil que se construyeron en los primeros años de la fundación del corregimiento. El segundo capítulo expone las particularidades del corregimiento desde los años 80 y destaca las situaciones de violencia que se vivieron allí hasta el momento del desplazamiento. Esta parte de la investigación indaga por las relaciones del Estado y la sociedad civil en el contexto de violencia. El último capítulo, se concentra en la población desplazada de Sabaneta y su relación con Buenavista, municipio receptor ubicado en el Departamento de Sucre. De acuerdo con el último objetivo, este capítulo muestra la relación de las personas en situación de desplazamiento con las entidades estatales.Sociólogo (a)Pregrad

Arabidopsis thaliana: A model host plant to study plant-pathogen interaction using Chilean field isolates of Botrytis cinerea

http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000200004&lng=es&nrm=isoOne of the fungal pathogens that causes more agriculture damage is Botrytis cinerea. Botrytis is a constant threat to crops because the fungus infects a wide range of host species, both native and cultivated. Furthermore, Botrytis persists on plant debris in and on the soil. Some of the most serious diseases caused by Botrytis include gray mold on vegetables and fruits, such as grapes and strawberries. Botrytis also causes secondary soft rot of fruits and vegetables during storage, transit and at the market. In many plant-pathogen interactions, resistance often is associated with the deposition of callose, accumulation of autofluorescent compounds, the synthesis and accumulation of salicylic acid as well as pathogenesis-related proteins. Arabidopsis thaliana has been used as a plant model to study plant-pathogen interaction. The genome of Arabidopsis has been completely sequenced and this plant serves as a good genetic and molecular model. In this study, we demonstrate that Chilean field isolates infect Arabidopsis thaliana and that Arabidopsis subsequently activates several defense response mechanisms associated with a hypersensitive response. Furthermore, we propose that Arabidopsis may be used as a model host species to analyze the diversity associated with infectivity among populations of Botrytis cinerea field isolates

Transcription in vitro of ø29 DNA and EcoRI fragments by Bacillus subtilis RNA polymerase

EcoRI fragments A, B and C produced from linear φ29 DNA, but not D or E fragments, are transcribed by purified Bacillus subtilis RNA polymerase. The transcription of fragments A and C is initiated preferentially with GTP and to a lesser extent with ATP; the reverse happens in the case of fragment B. The dinucleotides GpU and GpA respectively, compete specifically with the incorporation of [γ-32P]GTP directed by fragments A and C. The RNA synthesized in vitro by purified B. subtilis RNA polymerase is highly asymmetric. Most of the RNA synthesis directed by fragments A and C is early RNA. However, most of the RNA produced by fragment B is anti-late-RNA. Addition of crude extracts inhibit the transcription of fragment B but not that of fragments A and C.Comisión Asesora para el Desarrollo de la Investigación Científica y Técnica y Dirección General de SanidadPeer reviewe

In vitro transcription of the Bacillus subtilis phage ø29 DNA by Bacillus subtilis and Escherichia coli RNA polymerases

The Escherichia coli RNA polymerase bound to phage ø29 DNA has been visualized by electron microscopy. Thirteen specific binding sites have been observed at 1.7,2.6,5.5,10.4,13.7,25.2,25.7,26.3,33.5,59.5,69.2,91.7 and 99.6 DNA length units and they have been named A1,A1I,A1II,A1III,A1IV,A2,A2I,A3,A4,B1,B1I,C1 and C2, respectively. The binding sites A1,A2,A3,B1,C1 and C2 coincide with those found with Bacillus subtills RNA polymerase. The transcription of phage ø29 DNA with B. subtilis or E. coli RNA polymerases has been studied. With the B. subtilis RNA polymerase eight transcripts were found, starting at positions corresponding to the binding sites A1, A1III, A2,A3,B1I,B2 and C2, respectively. With the E. coli RNA polymerase the same transcripts were found and a new one starting at position corresponding to the A4 binding site. The RNAs starting at binding sites A1,A1III,A2,B1I, B2,C1 and C2 are transcribed from right to left, as expected for early RNA. The RNAs which initiate at positions A3 arid A4 are transcribed from left to right and probably correspond to late RNAs.Peer reviewe

A new protein domain for binding to DNA through the minor groove

PMID:7925279Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative alpha-helix. DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained. A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant. Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment. This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove. In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA. All these results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove.This work has been supported by grants SROI GM27242-15 from the National Institutes of Health, BIOT-CT91-0268 and CHRX-CT92-0010 from the European Economic Community and PB90/0091 from the Direcci6n General de Investigaci6n Cientffica Tecnica. The institutional help of Fundaci6n Ram6n Areces is also acknowledged. R.Freire was a recipient of a predoctoral fellowship from Comunidad Autonoma de MadridPeer reviewe