Internal initiation of translation of the TrkB mRNA is mediated by multiple regions within the 5′ leader

Translational regulation of the dendritically localized mRNA encoding for the neurotrophin receptor TrkB has important ramifications for synaptic function. We examined whether the TrkB mRNA is translated through an internal initiation entry site (IRES). The human TrkB 5′ leaders are derived from the use of alternative promoters and alternative splicing, but all 5′ leaders share a common exon. Insertion of a full-length 5′ leader, as well as the common exon into the intercistronic region of a dicistronic luciferase construct, yielded luciferase activity generated from the second cistron that was either equivalent or higher than that observed from the encephalomyocarditis virus IRES. Moreover, inhibiting cap-dependent translation ex vivo and in in vitro lysates had only a minimal effect on the translation of mRNA containing the TrkB 5′ leader. Dissecting the 5′ leader showed that the IRES is located in the exon common to all TrkB 5′ leaders. Moreover, six regions ranging from 2 to 25 nt were identified that either promoted or inhibited IRES activity. Taken together, these results suggest that the 5′ leader of the human TrkB mRNA contains multiple cis-elements that regulate internal initiation of translation and that this mechanism may contribute significantly to the translation of the TrkB mRNA in neuronal dendrites

Space-time variability of the pair AS-CD in the hydric resource and risk zones at the southwestern section of the Pampean Plain, Argentine

En el recurso hídrico de la llanura pampeana, el As se encuentra entre los contaminantes químicos naturales más relevantes y coexiste con otros elementos traza. Estudios precedentes han postulado que el riesgo potencial representado por el As se vería reforzado por su asociación hidrogeoquímica con el Cd, cuando ambas concentraciones superan los límites de tolerancia establecidos por la Organización Mundial de la Salud (OMS). Según el grado de severidad de la afectación pueden evolucionar enfermedades cardiovasculares, hipertensión, problemas de reproducción y anomalías en los huesos, etc. Sobre la base de lo expuesto se establecen los siguientes objetivos: 1- estudiar la variación espacio-temporal de las concentraciones de As-Cd, 2- delimitar las áreas de mayor riesgo para la comunidad, 3- analizar el grado de correlación entre ellos y 4- evaluar las probables fuentes de aporte. El estudio se realizó en la cuenca del arroyo El Divisorio (Provincia de Buenos Aires), cuyo curso principal desemboca en el embalse Paso de las Piedras, que es utilizado para abastecer de agua potable a una población >400.000 habitantes. Con la finalidad de evaluar la calidad de las aguas se realizaron tres campañas de muestreos en distintos períodos estacionales: invierno de 2008, otoño de 2009 y primavera-verano de 2009. La toma de muestras se llevó a cabo en perforaciones (38) y en el recurso hídrico superficial (9). Las determinaciones cuali-cuantitativas de las muestras de agua fueron realizadas por el Laboratorio de Análisis Químicos (LANAQUI) del CERZOS-CONICET (UNS). Se confeccionaron mapas de isoconcentraciones, los que indican los sectores más afectados, además de realizar análisis de correlaciones, regresiones lineales y análisis de frecuencia. La distribución espacio-temporal de las concentraciones de Cd en las aguas subterráneas no presentaron diferencias y el valor guía de referencia fue superado en el 36% de las muestras, observándose un parche de alto riesgo para el consumo humano en el sudoeste. En los tramos bajos de la cuenca, se hallaron los niveles máximos de As y el 86 % de las muestras analizadas superan los niveles guía. Para las aguas superficiales, el arroyo presenta concentraciones de Cd crecientes hacia la desembocadura, superando el límite de referencia el 37,5 % de los puntos muestreados. En el mismo período de tiempo y en cercanías a la desembocadura, el As presentó las máximas magnitudes. Las concentraciones no aceptables de Cd se correlacionaron significativamente con altos niveles de As, acrecentando la peligrosidad de la ingesta en la población residente. La presencia de Cd obedece a fuentes naturales vinculadas a las litologías de origen eólico y de tipo loéssico, las que se hallan en contacto con las aguas superficiales y constituyen las secuencias sedimentarias en la que se hospeda el acuífero freático.Arsenic is among the most relevant natural contaminants and it coexists with other oligoelements in the hydric resource of the pampean plain of Argentine. Antecedent studies have postulated that the potential risk of As would be strengthened by the hydrogeochemical association with Cd, when their concentrations exceed the guide values settled by the World Health Organization (WHO). Depending on the degree of severity of water quality affection, cardiovascular diseases, hypertension, reproductive problems and bone abnormalities may develop. On this basis the following objectives were proposed in our research: 1- to study the space-time variability of the As-Cd concentrations, 2- to delimit the areas of the higher risk for the community, 3- to analyze their degree of correlation and 4- to evaluate the probable provision sources. The study was developed in the Aº. El Divisorio catchment (Buenos Aires province), whose principal course discharges into Paso de las Piedras dam, which is used to source tapped water to a population of >400,000 inhabitants. Three sampling campaings were developed during different seasonal periods: winter 2008, autumm 2009 and spring-season 2009. For water quality evaluation samples were collected at water wells (n: 38) and surface waters (n: 9). Quali-Quantitative analysis were performed at the Laboratorio de Análisis Químicos (LANAQUI) of CERZOS-CONICET (UNS). Maps of isoconcentrations were produced with the concentrations values using the Surfer V.8 software. The statistical analysis for correlation analysis, linear regressions and frequency analysis was performed applying the InfoStat software with a design of repeated measures. The space and time Cd concentrations do not show differences and in 36% of the groundwater samples exceeded the guide value, showing a patch of a high risk for human consumption as drinking water at southwestern areas. High arsenic levels surpassed the guide values in 86% of the sampled groundwaters with the highest levels in the low landforms of the studied basin. Towards the river mouth, the surface waters yielded increased Cd levels exceeding the reference level in 37.5% of samples. At this position and during the same period, As had the highest concentrations. Unacceptable Cd concentrations had a significant correlation with high As levels, increasing the risk for human consumption as drinking water. The presence of As-Cd is related to natural sources and appears linked to the aeolian loess lithologies contacting surface and groundwaters in the sedimentary sequences.Asociación Argentina de Geofísicos y Geodesta

Opening-closing dynamics of the mitochondrial transcription pre-initiation complex

Promoter recognition and local melting of DNA are key steps of transcription initiation catalyzed by RNA polymerase and initiation factors. From single molecule fluorescence resonance energy transfer studies of the yeast (Saccharomyces cerevisiae) mitochondrial RNA polymerase Rpo41 and its transcription factor Mtf1, we show that the pre-initiation complex is highly dynamic and undergoes repetitive opening-closing transitions that are modulated by Mtf1 and ATP. We found that Rpo41 alone has the intrinsic ability to bend the promoter but only very briefly. Mtf1 enhances bending/opening transition and suppresses closing transition, indicating its dual roles of nucleating promoter opening and stabilizing the open state. The cognate initiating ATP prolongs the lifetime of the open state, plausibly explaining the 'ATP sensing mechanism' suggested for the system. We discovered short-lived opening trials upon initial binding of Rpo41-Mtf1 before the establishment of the opening/closing equilibrium, which may aid in promoter selection before the formation of stable pre-initiation complex. The dynamics of open complex formation provides unique insights into the interplay between RNA polymerase and transcription factors in regulating initiation.open4

FilmArray, an Automated Nested Multiplex PCR System for Multi-Pathogen Detection: Development and Application to Respiratory Tract Infection

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the “FilmArray”, which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon

Identification and characterization of the mitochondrial RNA polymerase and transcription factor in the fission yeast Schizosaccharomyces pombe

We have characterized the mitochondrial transcription factor (Mtf1) and RNA polymerase (Rpo41) of Schizosaccharomyces pombe. Deletion mutants show Mtf1 or Rpo41 to be essential for cell growth, cell morphology and mitochondrial membrane potential. Overexpression of Mtf1 and Rpo41 can induce mitochondrial transcription. Mtf1 and Rpo41 can bind and transcribe mitochondrial promoters in vitro and the initiating nucleotides were the same in vivo and in vitro. Mtf1 is required for efficient transcription. We discuss the functional differences between Mtf1 and Rpo41 of S. pombe with Saccharomyces cerevisiae and higher organisms. In contrast to S. cerevisiae, the established model for mitochondrial transcription, S. pombe, a petite-negative yeast, resembles higher organisms that cannot tolerate the loss of mitochondrial function. The S. pombe and human mitochondrial genomes are similar in size and much smaller than that of S. cerevisiae. This is an important first step in the development of S. pombe as an alternative and complementary model system for molecular genetic and biochemical studies of mitochondrial transcription and mitochondrial–nuclear interactions. This is the first systematic study of the cellular function and biochemistry of Rpo41 and Mtf1 in S. pombe

Molecular architecture of a dynamin adaptor: implications for assembly of mitochondrial fission complexes

Structural and functional studies of the Mdv1 dimer reveal how this mitochondria-associated adaptor protein orients and stabilizes the assembly of dynamins on membranes to promote mitochondrial fission

A novel function of the mitochondrial transcription factor Mtf1 in fission yeast; Mtf1 regulates the nuclear transcription of srk1

In eukaryotic cells, Mtf1 and its homologues function as mitochondrial transcription factors for the mitochondrial RNA polymerase in the mitochondrion. Here we show that in fission yeast Mtf1 exerts a non-mitochondrial function as a nuclear factor that regulates transcription of srk1, which is a kinase involved in the stress response and cell cycle progression. We first found Mtf1 expression in the nucleus. A ChIP-chip approach identified srk1 as a putative Mtf1 target gene. Over expression of Mtf1 induced transcription of the srk1 gene and Mtf1 deletion led to a reduction in transcription of the srk1 gene in vivo. Mtf1 overexpression causes cell elongation in a srk1 dependent manner. Mtf1 overexpression can cause cytoplasmic accumulation of Cdc25. We also provide biochemical evidence that Mtf1 binds to the upstream sequence of srk1. This is the first evidence that a mitochondrial transcription factor Mtf1 can regulate a nuclear gene. Mtf1 may also have a role in cell cycle progression

Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

The Parkin ubiquitin ligase marks the mitofusins Mfn1 and Mfn2 for proteasome-dependent degradation, promoting disposal of damaged mitochondria by preventing their fusion with healthy organelles

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages