The characterization of an epithelial chloride channel and purification of a channel component

A membrane fraction (M$\sb{\rm PS}$), enriched in Cl$\sp-$ channels, has been isolated from bovine tracheal epithelia and renal cortex homogenates by hydrophobic chromatography. The tracheal fraction shows a 37 fold enrichment of Cl$\sp-$ channels over crude tracheal homogenates by net Cl$\sp-$ measurements in membrane vesicles. Alkaline phosphatase and (Na$\sp+$ + K$\sp+$)-ATPase are not found in these membranes, suggesting that they are not apical or basolateral plasma membranes. The M$\sb{\rm PS}$ fraction exhibits a protein profile unlike that of other membrane fractions with major proteins of 200 kDa and 42 kDa, proteins of 30 to 35 kDa, and lesser amounts of other proteins. Reconstitution of M$\sb{\rm PS}$ fractions from both trachea and kidney into planar lipid bilayers demonstrates the presence of a single type of anion channel. The current-voltage relationship of this channel is linear with a slope conductance of 84 pS in symmetrical 400 mM KCl, and is identical to that of the predominant anion channel observed in tracheal apical membranes under similar conditions (Valdivia, Dubinsky, and Coronado. Science, 1988). In addition, the voltage dependence, selectivity sequence of Cl$\sp- >$ Br$\sp- \ge$ I$\sp-$, and inhibition by low concentrations of the Cl$\sp-$ channel blocker, DIDS, correspond to those of the predominant apical membrane channel. Thus, although the M$\sb{\rm PS}$ fraction appears to be of subcellular origin, it may be functionally related to an apical membrane Cl$\sp-$ permeability. When renal M$\sb{\rm PS}$ membranes were treated with the detergent octyl-glucoside (OG, 2%) and centrifuged, the supernatant, sM$\sb{\rm PS}$, showed a 2 to 7-fold enrichment in specific Cl$\sp-$ flux activity compared with the detergent treated M$\sb{\rm PS}$. These solubilized proteins were then size fractionated on a Superose 12 HPLC gel filtration column, followed by fractionation on a Mono Q HPLC anion exchange column. Fractions that eluted in high salt consistently exhibited significant Cl$\sp-$ flux activity. These fractions had protein profiles consisting of a major band at 34 kDa, a band at 66 kDa, and variable faint bands. Fractions eluting in lower salt had protein profiles consisting of a single band at 34 kDa, and often had little or no Cl$\sp-$ flux activity. However, co-reconstitution of the low salt, solely 34 kDa protein-containing Mono Q fractions with sM$\sb{\rm PS}$ resulted in an enhancement of flux activity compared to that of sM$\sb{\rm PS}$ reconstituted alone. Flux assays of active Mono Q fractions showed that the channel retained its DIDS sensitivity. Applying sM$\sb{\rm PS}$ to a DIDS-affinity column and eluting with salt resulted in fractions with protein profiles again consisting of at least one major band at 34 kDa, a band at 66 kDa, and variable faint bands. Co-reconstitution with sM$\sb{\rm PS}$ again resulted in an enhancement of activity. Thus, the 34 kDa protein appears to be a component of the M$\sb{\rm PS}$ Cl$\sp-$ channel. ^

Alcohol consumption and breast cancer risk among Hispanic and non-Hispanic women in New Mexico

Breast cancer incidence and mortality rates for Hispanic women are lower than for non-Hispanic white (NHW) women, but recently rates have increased more rapidly among Hispanic women. Many studies have shown a consistent increased breast cancer risk associated with modest or high alcohol intake, but few included Hispanic women. Alcohol consumption and risk of breast cancer was investigated in a New Mexico statewide population-based case-control study. The New Mexico Tumor Registry ascertained women, newly diagnosed with breast cancer (1992–1994) aged 30–74 years. Controls were identified by random digit dialing and were frequency-matched for ethnicity, age-group, and health planning district. In-person interviews of 712 cases and 844 controls were conducted. Data were collected for breast cancer risk factors, including alcohol intake. Recent alcohol intake data was collected for a four-week period, six months prior to interview. Past alcohol intake included information on alcohol consumption at ages 25, 35, and 50. History of alcohol consumption was reported by 81% of cases and 85% of controls. Of these women, 42% of cases and 48% of controls reported recent alcohol intake. Results for past alcohol intake did not show any trend with breast cancer risk, and were nonsignificant. Multivariate-adjusted odds ratios for recent alcohol intake and breast cancer suggested an increased risk at the highest level for both ethnic groups, but estimates were unstable and statistically nonsignificant. Low level of recent alcohol intake (<148 grams/week) was associated with a reduced risk for NHW women (Odds Ratio (OR) = 0.49 95% Confidence Interval (CI) 0.35–0.69). This pattern was independent of hormone-receptor status. The reduced breast cancer risk for low alcohol intake was present for premenopausal (OR = 0.29, 95% CI 0.15–0.56) and postmenopausal NHW women (OR = 0.56, 95% CI 0.35–0.90). The possibility of an increased risk associated with high alcohol intake could not be adequately addressed, because there were few drinkers with more than light to moderate intake, especially among Hispanic women. An alcohol-estrogen link is hypothesized to be the mechanism responsible for increased breast cancer risk, but has not been consistently substantiated. More studies are needed of the underlying mechanism for an association between alcohol intake and breast cancer. ^

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we focus on the functional characterisation of an 18 nucleotide long ncRNA candidate derived from an open reading frame (ORF) of an annotated S. cerevisiae gene, which encodes a tRNA methyltransferase. Yeast cells lacking this tRNA methyltransferase showed clear growth defects in high salt containing media. Genetic analysis showed that the absence of the mRNA-derived ncRNA rather than the absence of the tRNA methyltransferase activity is responsible for the observed phenotype. Since we performed a screen for small ribosome-associated RNAs we examined the regulatory potential of the synthetic 18mer during translation in vitro and in vivo. Metabolic labeling experiments in the presence of the synthetic 18mer RNA revealed an inhibitory potential on the global protein biosynthesis rate. In vitro translation and northern blot analysis further strengthen the hypothesis, that this RNA is a ribosome-associated regulatory ncRNA. Our studies in pro- and eukaryotic model organisms reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

Natural antisense transcripts of Trifolium repens dehydrins

The recently described complex nature of some dehydrin-coding sequences in Trifolium repens could explain the considerable variability among transcripts originating from a single gene.1 For some of the sequences the existence of natural antisense transcripts (NAT s), which could form sense-antisense (SAS) pairs, was predicted. The present study demonstrates that cis-natural antisense transcripts of 2 dehydrin types (YnKn and YnSKn) accumulate in white clover plants subjected to treatments with polyethylene glycol (PEG), abscisic acid (ABA), and high salt concentration. The isolated YnKn cis-NAT s mapped to sequence site enriched in alternative start codons. Some of the sense-antisense pairs exhibited inverse expression with differing profiles which depended on the applied stress. A natural antisense transcript coding for an ABC F family protein (a trans-NAT) which shares short sequence homology with YnSKn dehydrin was identified in plants subjected to salt stress. Forthcoming experiments will evaluate the impact of NAT s on transcript abundances, elucidating the role of transcriptional and post-transcriptional interferences in the regulation of dehydrin levels under various abiotic stresses.

Diversity of morphological patterns in supramolecular polymers formed from the amphiphilic oligomers of pyrenes

Very important aspects of the modern nanotechnology are control and prediction of arraying patterns of opto- and electroactive molecules in discrete objects on nanoscale level both on surface and solution. Consequqntly, a self-assembly of small molucules provides such an opportunity.For example, oligopyrenotides (OPs, short amphiphilic pyrene oligomers) represent a novel class of amphiphilic molecules which tend to aggegate in aqueous phase. As has been already shown, OPs are able to form 1D supramolecular polymer only under high salt concentration. Since programmed arraying of polyaromatic hydrocarbons in structurally defined objects could offer enhanced performance over the individual components, prediction and controlling of their spatial arrangement remains challenging. Herein we demonstrate that substitution type of the pyrene is crutial, and it determines a morphology of the assemblies. Thus, a 1.6-linkage causes a formation of large, free-standing 2D supromolecular polymers with a thickness 2 nm. These assemblies possess a high degree of an internal order: the interior consists of hydrophobic pyrenes and alkyl chains, whereas the exterior exists as a net of hydrophilic negatively charged phosphates. Contrary, a 1.8-linkage exclusiveley leads to a formation of long (up to a few micrometer), nanometer thick helical supramolecular polymers. These structures tend to form even more complex structures (bundles, superhelixes). Moreover for both molecules, the polymerizations occurs via a nucleation-elongation mechanism. To study Py3 self-assembly, we carried out whole set of spectroscopic (UV/vis, fluorescence, DLS) and microscopic experiments (AFM).

Colon-specific deletion of epithelial sodium channel causes sodium loss and aldosterone resistance

Aldosterone promotes electrogenic sodium reabsorption through the amiloride-sensitive epithelial sodium channel (ENaC). Here, we investigated the importance of ENaC and its positive regulator channel-activating protease 1 (CAP1/Prss8) in colon. Mice lacking the αENaC subunit in colonic superficial cells (Scnn1a(KO)) were viable, without fetal or perinatal lethality. Control mice fed a regular or low-salt diet had a significantly higher amiloride-sensitive rectal potential difference (∆PDamil) than control mice fed a high-salt diet. In Scnn1a(KO) mice, however, this salt restriction-induced increase in ∆PDamil did not occur, and the circadian rhythm of ∆PDamil was blunted. Plasma and urinary sodium and potassium did not change with regular or high-salt diets or potassium loading in control or Scnn1a(KO) mice. However, Scnn1a(KO) mice fed a low-salt diet lost significant amounts of sodium in their feces and exhibited high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAP1/Prss8 in colonic superficial cells (Prss8(KO)) were viable, without fetal or perinatal lethality. Compared with controls, Prss8(KO) mice fed regular or low-salt diets exhibited significantly reduced ∆PDamil in the afternoon, but the circadian rhythm was maintained. Prss8(KO) mice fed a low-salt diet also exhibited sodium loss through feces and higher plasma aldosterone levels. Thus, we identified CAP1/Prss8 as an in vivo regulator of ENaC in colon. We conclude that, under salt restriction, activation of the renin-angiotensin-aldosterone system in the kidney compensated for the absence of ENaC in colonic surface epithelium, leading to colon-specific pseudohypoaldosteronism type 1 with mineralocorticoid resistance without evidence of impaired potassium balance.

Sugar- and artificially sweetened beverages and intrahepatic fat: A randomized controlled trial.

OBJECTIVE To test the hypothesis that substituting artificially sweetened beverages (ASB) for sugar-sweetened beverages (SSB) decreases intrahepatocellular lipid concentrations (IHCL) in overweight subjects with high SSB consumption. METHODS About 31 healthy subjects with BMI greater than 25 kg/m(2) and a daily consumption of at least 660 ml SSB were randomized to a 12-week intervention in which they replaced SSBs with ASBs. Their IHCL (magnetic resonance spectroscopy), visceral adipose tissue volume (VAT; magnetic resonance imaging), food intake (2-day food records), and fasting blood concentrations of metabolic markers were measured after a 4-week run-in period and after a 12-week period with ASB or control (CTRL). RESULTS About 27 subjects completed the study. IHCL was reduced to 74% of the initial values with ASB (N = 14; P < 0.05) but did not change with CTRL. The decrease in IHCL attained with ASB was more important in subjects with IHCL greater than 60 mmol/l than in subjects with low IHCL. ALT decreased significantly with SSB only in subjects with IHCL greater than 60 mmol/l. There was otherwise no significant effect of ASB on body weight, VAT, or metabolic markers. CONCLUSIONS In subjects with overweight or obesity and a high SSB intake, replacing SSB with ASB decreased intrahepatic fat over a 12-week period.

The cDNA cloning of murine HIP /L29 and the functional analysis of HIP/L29 domains

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is a heparin/heparan sulfate (Hp/HS) binding protein found in many adult human tissues. Potential functions of this protein are promotion of embryo adhesion, modulation of blood coagulation, and control of cell growth. While these activities are diverse, the ability of human HIP/L29 to interact with Hp/HS at the cell surface may be a unifying mechanism of action since Hp/HS influences all of these processes. A murine ortholog has been identified that has 78.8% homology over the entire sequence and identity over the N-terminal 64 amino acids when compared to human HIP/L29. Northern, Western, and immunohistochemical analysis shows that murine HIP/L29 mRNA and protein are expressed in a tissue specific manner. Murine HIP/L29 is enriched in the membrane fraction of NmuMG cells where it is eluted with high salt, suggesting that it is a peripheral membrane protein. The ability of murine HIP/L29 to bind Hp is verified by studies using native and recombinant forms of murine HIP/L29. A synthetic peptide (HIP peptide-2) derived from the identical N-terminal region of HIP/L29 proteins was tested for the ability to bind Hp and support cell adhesion. This peptide was chosen because it conforms to a proposed consensus sequence for Hp/HS binding peptides. HIP peptide-2 binds Hp in a dose-dependent, saturable, and selective manner and supports Hp-dependent cell adhesion. However, a scrambled form of this peptide displayed similar activities indicating a lack of peptide sequence specificity required for activity. Lastly, an unbiased approach was used to identify sequences within human and mouse HIP/L29 proteins necessary for Hp/HS binding. A panel of recombinant proteins was made that collectively are deficient in every human HIP/L29 domain. The activities of these deletion mutants and recombinant murine HIP/L29 were compared to the activity of recombinant human HIP/L29 in a number of assays designed to look at differences in the ability to bind Hp/HS. These studies suggest that each domain within human HIP/L29 is important for binding to Hp/HS and divergences in the C-terminus of human and mouse HIP/L29 account for a decrease in murine HIP/L29 affinity for Hp/HS. It is apparent that multiple domains within human and mouse HIP/L29 contribute to the function of Hp/HS binding. The interaction of multiple HIP/L29 domains with Hp/HS will influence the biological activity of HIP/L29 proteins. ^

Cartografía de QTL asociados a la tolerancia a estrés hídrico en frijol común (Phaseolus vulgaris L.)

Phaseolus vulgaris L. (frijol común o judía) es una leguminosa de gran demanda para la nutrición humana y un producto agrícola muy importante. Sin embargo, la producción de frijol se ve limitada por presiones ambientales como la sequía. En México, el 85% de la cosecha de frijol se produce en la temporada de primavera-verano, principalmente en las regiones del altiplano semiárido con una precipitación anual entre 250 y 400 mm. A pesar del implemento de tecnología en el campo, los factores naturales impiden al agricultor llegar a los rendimientos deseados. El Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), como instituto de investigación gubernamental en México, tiene como objetivo la mejora de cultivos estratégicos, uno de ellos, P. vulgaris. Los estudios en relación a la sequía se enfocan especialmente en la selección de genotipos tolerantes, los cuales son sometidos en condiciones de estrés y monitoreando parámetros como el rendimiento y peso de semilla, además de algunos indicadores tales como índice de cosecha. El resultado de estos trabajos ha sido la obtención de variedades con mayor tolerancia a la sequía, tales como Pinto Villa y Pinto Saltillo. En los últimos años se ha avanzado notablemente en el conocimiento de las bases moleculares en las respuestas de las plantas al estrés. De acuerdo a diversos estudios se ha demostrado que las plantas bajo estrés por sequía experimentan cambios en la expresión de genes involucrados en la señalización, regulación de la transcripción y la traducción, transporte de agua y la función directa en la protección celular. También se ha observado que el déficit de agua es causado por las temperaturas extremas y la alta concentración de sales, por lo que al nivel molecular, las respuestas al estrés tienen puntos de especificidad y puntos de entrecruzamiento. La sequía puede generar estreses secundarios, tales como el nutricional, oxidativo y osmótico. Sin embargo, es necesario identificar y caracterizar muchos de los componentes involucrados en las respuestas al déficit hídrico, la caracterización de estos genes permitirá tener una mejor comprensión de los mecanismos bioquímicos y fisiológicos involucrados en la tolerancia al estrés. Actualmente, con el apoyo de la biología molecular se han identificado algunos genes que otorgan ventajas para la adaptación a ambientes desfavorables. Por lo que el objetivo del presente trabajo es identificar marcadores genéticos asociados a rasgos fenotípicos con énfasis a la tolerancia a estrés hídrico en P. vulgaris. Una vez establecidos los marcadores asociados al estrés hídrico, es factible considerar su uso para la selección asistida por marcadores en líneas o variedades de frijol de interés para los mejoradores. Se evaluaron 282 familias F3:5 derivadas de la cruza entre los cultivares Pinto Villa y Pinto Saltillo. Las familias se sembraron bajo un diseño simple de látice 17x17, el experimento se llevo acabo en el ciclo primavera-verano del 2010 y 2011, y otoñoinvierno de 2010 en el Campo Experimental Bajío del INIFAP con dos repeticiones para cada tratamiento de humedad (riego completo y sequía terminal). En todos los genotipos se realizó el fenotipado (variables fenotípicas) y el genotipado a través de marcadores moleculares. Los análisis estadísticos se basaron en el análisis de componentes principales (Eigen Analysis Selection Index Method, ESIM), la asociación entre marcadores SNP y el fenotipado (paquete SNPassoc para R) y el análisis de varianza (ANOVA). Los valores ESIM mostraron que las variables de Rendimiento, Días a floración, Días a madurez fisiológica e Índice de cosecha fueron sobresalientes en sequía terminal, por lo que se sugieren tomarse en consideración para los estudios de sequía en P. vulgaris como monitores de evaluación a la resistencia. Se identificaron nueve familias sobresalieron por sus valores ESIM (PV/PS6, 22, 131, 137, 149, 154, 201, 236 y 273), además de presentar valores superiores para el rendimiento en comparación con los parentales. Estos genotipos son candidatos interesantes para realizar estudios de identificación de loci asociados con la respuesta al estrés, y como potenciales parentales en el desarrollo de nuevas variedades de frijol. En los análisis de asociación SNPassoc se identificaron 83 SNPs significativos (p<0,0003) asociados a los rasgos fenotípicos, obteniendo un total de 222 asociaciones, de las cuales predomina el modelo genético de codominancia para las variables Días a floración, Periodo reproductivo y Biomasa total. Treinta y siete SNPs se identificaron a diferentes funciones biológicas a través del análisis de anotación funcional, de los cuales 12 SNPs (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 y 142) sobresalen por su asociación al fenotipado, y cuya anotación funcional indica que se encuentran en genes relacionados a la tolerancia a la sequía, tales como la actividad kinasa, actividad metabólica del almidón, carbohidratos y prolina, respuesta al estrés oxidativo, así como en los genes LEA y posibles factores de transcripción. En el caso de los análisis ANOVA, se identificaron 72 asociaciones entre los SNPs y las variables fenotípicas (F< 3,94E-04). Las 72 asociaciones corresponden a 30 SNPs y 7 variables fenotípicas, de las que predomina Peso de 100 semillas y Periodo reproductivo. Para los rasgos de Rendimiento, Índice de cosecha y Días a madurez fisiológica se presentaron asociaciones con seis SNPs (17, 34, 37, 50, 93 y 107), de los cuales, a los SNP37 y SNP107 fueron identificados a la anotación biológica de protein binding. Por otro lado, los SNP106 y SNP128 asociados al Periodo reproductivo, son genes con actividad kinasa y actividad metabólica del almidón, respectivamente. Para los marcadores tipo AFLP, se identificaron 271 asociaciones (F<2,34E-04). Las asociaciones corresponden a 86 AFLPs con todas las variables fenotípicas evaluadas, de las que predomina peso de 100 semillas, Días a floración y Periodo reproductivo. Debido a que los en los AFLPs no es posible determinar su anotación biológica, se proponen como marcadores potenciales relacionados a la resistencia a la sequía en frijol. Los AFLPs candidatos requieren más estudios tales como la secuenciación de los alelos respectivos, así como la identificación de éstas secuencias en el genoma de referencia y su anotación biológica, entre otros análisis, de esta manera podríamos establecer aquellos marcadores candidatos a la validación para la selección asistida. El presente trabajo propone tanto genotipos como marcadores genéticos, que deben ser validados para ser utilizados en el programa de mejoramiento de P. vulgaris, con el objetivo de desarrollar nuevas líneas o variedades tolerantes a la sequía. ABSTRACT Phaseolus vulgaris L. (common bean or judia) is a legume of great demand for human consumption and an important agricultural product. However, the common bean production is limited by environmental stresses, such as drought. In Mexico, 85% of the common bean crop is produced in the spring-summer season mainly in semiarid highland regions with a rainfall between 250 and 400 mm per year. In spite of the improvement of crop technology, the natural factors hamper getting an optimal yield. The National Institute for Forestry, Agriculture and Livestock (INIFAP) is a government research institute from Mexico, whose main objective is the genetic breeding of strategic crops, like P. vulgaris L. The drought tolerance studies particularly focus on the selection of bean tolerant genotypes, which are subjected to stress conditions, by means of monitoring parameters such as yield and seed weight, plus some agronomic indicators such as harvest index. The results of these works have led to obtain cultivars with higher drought tolerance such as Pinto Villa and Pinto Saltillo. Significant achievements have been recently made in understanding the molecular basis of stress plant responses. Several studies have shown that plants under drought stress present changes in gene expression related to cell signalling, transcriptional and translational regulation, water transport and cell protection. In addition, it has been observed that the extreme temperatures and high salt concentrations can cause a water deficiency so, at the molecular level, stress responses have specific and crossover points. The drought can cause secondary stresses, such as nutritional, oxidative and osmotic stress. It is required the identification of more components involved in the response to water deficit, the characterization of these genes will allow a better understanding of the biochemical and physiological mechanisms involved in stress tolerance. Currently, with the support of molecular biology techniques, some genes that confer an advantage for the crop adaptation to unfavourable environments have been identified. The objective of this study is to identify genetic markers associated with phenotypic traits with emphasis on water stress tolerance in P. vulgaris. The establishment of molecular markers linked to drought tolerance would make possible their use for marker-assisted selection in bean breeding programs. Two hundred and eighty two F3:5 families derived from a cross between the drought resistant cultivars Pinto Villa and Pinto Saltillo were evaluated. The families were sowed under a 17x17 simple lattice design. The experiment was conducted between spring-summer seasons in 2010 and 2011, and autumn-winter seasons in 2010 at the Bajio Experimental Station of INIFAP with two treatments (full irrigation and terminal drought). All families were phenotyped and genotyped using molecular markers. Statistical analysis was based on principal component analysis (Eigen Analysis Selection Index Method, ESIM), association analysis between SNP markers and phenotype (SNPassoc package R) and analysis of variance (ANOVA). The ESIM values showed that seed yield, days to flowering, days to physiological maturity and harvest index were outstanding traits in terminal drought treatment, so they could be considered as suitable parameters for drought-tolerance evaluation in P. vulgaris. Nine outstanding families for the ESIM values were identified (PV/PS6, 22, 131, 137, 149, 154, 201, 236 and 273), in addition, these families showed higher values for seed yield compared to the parental cultivars. These families are promising candidates for studies focused on the identification of loci associated to the stress response, and as potential parental cultivars for the development of new varieties of common bean. In the SNPassoc analysis, 83 SNPs were found significantly associated (p<0.0003) with phenotypic traits, obtaining a total of 222 associations, most of which involved the traits days to flowering, reproductive period and total biomass under a codominant genetic model. The functional annotation analysis showed 37 SNPs with different biological functions, 12 of them (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 and 142) stand out by their association to phenotype. The functional annotation suggested a connection with genes related to drought tolerance, such as kinase activity, starch, carbohydrates and proline metabolic processes, responses to oxidative stress, as well as LEA genes and putative transcription factors. In the ANOVA analysis, 72 associations between SNPs and phenotypic traits (F<3.94E- 04) were identified. All of these associations corresponded to 30 SNPs markers and seven phenotypic traits. Weight of 100 seeds and reproductive period were the traits with more associations. Seed yield, harvest index and days to physiological maturity were associated to six SNPs (17, 34, 37, 50, 93 and 107), the SNP37 and SNP107 were identified as located in protein binding genes. The SNP106 and SNP128 were associated with the reproductive period and belonged to genes with kinase activity and genes related to starch metabolic process, respectively. In the case of AFLP markers, 271 associations (F<2.34E-04) were identified. The associations involved 86 AFLPs and all phenotypic traits, being the most frequently associated weight of 100 seeds, days to flowering and reproductive period. Even though it is not possible to perform a functional annotation for AFLP markers, they are proposed as potential markers related to drought resistance in common bean. AFLPs candidates require additional studies such as the sequencing of the respective alleles, identification of these sequences in the reference genome and gene annotation, before their use in marker assisted selection. This work, although requires further validation, proposes both genotypes and genetic markers that could be used in breeding programs of P. vulgaris in order to develop new lines or cultivars with enhanced drought-tolerance.

Expression of Batis maritima methyl chloride transferase in Escherichia coli

Methyl chloride transferase, a novel enzyme found in several fungi, marine algae, and halophytic plants, is a biological catalyst responsible for the production of atmospheric methyl chloride. A previous paper reports the purification of this methylase from Batis maritima and the isolation of a cDNA clone of the gene for this enzyme. In this paper, we describe the isolation of a genomic clone of the methylase gene and the expression of recombinant methyl chloride transferase in Escherichia coli and compare the kinetic behavior of the wild-type and recombinant enzyme. The recombinant enzyme is active and promotes the production of methyl chloride by E. coli under in vivo conditions. The kinetic data indicate that the recombinant and wild-type enzymes have similar halide (Cl−, Br−, and I−)-binding capacities. Both the recombinant and wild-type enzymes were found to function well in high NaCl concentrations. This high salt tolerance resembles the activity of halobacterial enzymes rather than halophytic plant enzymes. These findings support the hypothesis that this enzyme functions in the control and regulation of the internal concentration of chloride ions in halophytic plant cells.

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Chrysanthemum chlorotic mottle viroid: Unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes

The causal agent of chrysanthemum chlorotic mottle (CChM) disease has been identified, cloned, and sequenced. It is a viroid RNA (CChMVd) of 398–399 nucleotides. In vitro transcripts with the complete CChMVd sequence were infectious and induced the typical symptoms of the CChM disease. CChMVd can form hammerhead structures in both polarity strands. Plus and minus monomeric CChMVd RNAs self-cleaved during in vitro transcription and after purification as predicted by these structures, which are stable and most probably act as single hammerhead structures as in peach latent mosaic viroid (PLMVd), but not in avocado sunblotch viroid (ASBVd). Moreover, the plus CChMVd hammerhead structure also appears to be active in vivo, because the 5′ terminus of the linear plus CChMVd RNA isolated from infected tissue is that predicted by the corresponding hammerhead ribozyme. Both hammerhead structures of CChMVd display some peculiarities: the plus self-cleaving domain has an unpaired A after the conserved A9 residue, and the minus one has an unusually long helix II. The most stable secondary structure predicted for CChMVd is a branched conformation that does not fulfill the rod-like or quasi-rod-like model proposed for the in vitro structure of most viroids with the exception of PLMVd, whose proposed secondary structure of lowest free energy also is branched. The unusual conformation of CChMVd and PLMVd is supported by their insolubility in 2 M LiCl, in contrast to ASBVd and a series of representative non-self-cleaving viroids that are soluble under the same high salt conditions. These results support the classification of self-cleaving viroids into two subgroups, one formed by ASBVd and the other one by PLMVd and CChMVd.

The Tail of a Yeast Class V Myosin, Myo2p, Functions as a Localization Domain

Myo2p is a yeast class V myosin that functions in membrane trafficking. To investigate the function of the carboxyl-terminal-tail domain of Myo2p, we have overexpressed this domain behind the regulatable GAL1 promoter (MYO2DN). Overexpression of the tail domain of Myo2p results in a dominant–negative phenotype that is phenotypically similar to a temperature-sensitive allele of myo2, myo2–66. The tail domain of Myo2p is sufficient for localization at low- expression levels and causes mislocalization of the endogenous Myo2p from sites of polarized cell growth. Subcellular fractionation of polarized, mechanically lysed yeast cells reveals that Myo2p is present predominantly in a 100,000 × g pellet. The Myo2p in this pellet is not solubilized by Mg++-ATP or Triton X-100, but is solubilized by high salt. Tail overexpression does not disrupt this fractionation pattern, nor do mutations in sec4, sec3, sec9, cdc42, or myo2. These results show that overexpression of the tail domain of Myo2p does not compete with the endogenous Myo2p for assembly into a pelletable structure, but does compete with the endogenous Myo2p for a factor that is necessary for localization to the bud tip.

Nascent Polypeptide–associated Complex Stimulates Protein Import into Yeast Mitochondria

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide–associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.