Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Autonomy,Equality, and Teaching among Aka Foragers and Ngandu Farmers of the Congo Basin

The significance of teaching to the evolution of human culture is under debate. We contribute to the discussion by using a quantitative, cross-cultural comparative approach to investigate the role of teaching in the lives of children in two small-scale societies: Aka foragers and Ngandu farmers of the Central African Republic. Focal follows with behavior coding were used to record social learning experiences of children aged 4 to 16 during daily life. “Teaching” was coded based on a functional definition from evolutionary biology. Frequencies, contexts, and subtypes of teaching as well as the identity of teachers were analyzed. Teaching was rare compared to observational learning, although both forms of social learning were negatively correlated with age. Children received teaching from a variety of individuals, and they also engaged in teaching. Several teaching types were observed, including instruction, negative feedback, and commands. Statistical differences in the distribution of teaching types and the identity of teachers corresponded with contrasting forager vs. farmer foundational cultural schema. For example, Aka children received less instruction, which empirically limits autonomous learning, and were as likely to receive instruction and negative feedback from other children as they were from adults. Commands, however, exhibited a different pattern suggesting a more complex role for this teaching type. Although consistent with claims that teaching is relatively rare in small-scale societies, this evidence supports the conclusion that teaching is a universal, early emerging cognitive ability in humans. However, culture (e.g., values for autonomy and egalitarianism) structures the nature of teaching.     

Physical mapping of DNA markers linked to stem rust resistance gene <Emphasis Type="Italic">Sr47</Emphasis> in durum wheat

Key message

Markers linked to stem rust resistance gene Sr47 were physically mapped in three small Aegilops speltoides chromosomal bins. Five markers, including two PCR-based SNP markers, were validated for marker-assisted selection.

Abstract

In durum wheat (Triticum turgidum subsp. durum), the gene Sr47 derived from Aegilops speltoides conditions resistance to race TTKSK (Ug99) of the stem rust pathogen (Puccinia graminis f. sp. tritici). Sr47 is carried on small interstitial translocation chromosomes (Ti2BL-2SL-2BL·2BS) in which the Ae. speltoides chromosome 2S segments are divided into four bins in genetic stocks RWG35, RWG36, and RWG37. Our objective was to physically map molecular markers to bins and to determine if any of the molecular markers would be useful in marker-assisted selection (MAS). Durum cultivar Joppa was used as the recurrent parent to produce three BC₂F₂ populations. Each BC₂F₂ plant was genotyped with markers to detect the segment carrying Sr47, and stem rust testing of BC₂F₃ progeny with race TTKSK confirmed the genotyping. Forty-nine markers from published sources, four new SSR markers, and five new STARP (semi-thermal asymmetric reverse PCR) markers, were evaluated in BC₂F₂ populations for assignment of markers to bins. Sr47 was mapped to bin 3 along with 13 markers. No markers were assigned to bin 1; however, 7 and 13 markers were assigned to bins 2 and 4, respectively. Markers Xrwgs38a, Xmag1729, Xwmc41, Xtnac3119, Xrwgsnp1, and Xrwgsnp4 were found to be useful for MAS of Sr47. However, STARP markers Xrwgsnp1 and Xrwgsnp4 can be used in gel-free systems, and are the preferred markers for high-throughput MAS. The physical mapping data from this study will also be useful for pyramiding Sr47 with other Sr genes on chromosome 2B.

     

Determination of zeranol and its metabolites in bovine muscle and liver by a chemiluminescence enzyme immunoassay: compared to an ultraperformance liquid chromatography tandem mass spectroscopy method

A chemiluminescent enzyme immunoassay (CLEIA) was compared to an ultraperformance liquid chromatography tandem mass spectroscopy (UPLC‐MS/MS) procedure for the analysis of zeranol and its metabolites in bovine tissue samples. Apparent recoveries from fortified samples by both methods were comparable at 0.5–4.0 µg/kg and a significant correlation was obtained. For CLEIA analysis, hapten mimicking the analyte was first synthesized and conjugated with the carrier protein bovine serum albumin as the immunogen to produce monoclonal antibody. The obtained antibody showed extensive cross‐reactivity toward zeranol metabolites (zearalanone). The limit of detection of CLEIA and UPLC‐MS/MS was 0.05 µg/kg and 0.5 µg/kg, respectively. Recoveries of both methods for fortified samples were higher than 75.0% with the coefficient of variation less than 15%. These results indicated that the combination of screening with CLEIA and confirmation with UPLC‐MS/MS for zeranol and its metabolites would be a reliable method for a large number of bovine samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target <Emphasis Type="Italic">lox</Emphasis>-Sites

Cre/lox recombination in vivo has become an important tool to induce chromosomal rearrangements like deletions. Using a combination of Ds transposition and Cre/lox recombination in two independent experiments on chromosomes 6 and 7 of tomato, two sets of somatic deletions up to a size of 200 kb were obtained. The efficiency of somatic deletion decreased with increasing deletion size. The largest germinally transmitted deletion had a size of only 55 kb. The results show that Cre-mediated deletion in somatic cells is less efficient when the lox sites are separated over larger distances. A further drop of the deletion efficiency after germinal transmission of the larger deletions can be explained by the probable loss of genes that are of vital importance to gametophyte function. Plasmid rescue of an 8.4 kb circularised deleted DNA showed that the Cre-mediated deletion takes place in tomato as expected. Since the circular Cre-deleted DNA could only be PCR amplified in plant cells where the deletion was not complete, the double-stranded DNA circle is assumed to be instable.     

Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases

Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence.     

PQN and DQN: algorithms for expression microarrays

An ideal expression algorithm should be able to tell truly different expression levels with small false positive errors and be robust to assay changes. We propose two algorithms. PQN is the non-central trimmed mean of perfect match intensities with quantile normalization. DQN is the non-central trimmed mean of differences between perfect match and mismatch intensities with quantile normalization. The quantiles for normalization can be either empirical or theoretical. When array types and/or assay change in a study, the normalization to common quantiles at the probe set level is essential. We compared DQN, PQN, RMA, GCRMA, DCHIP, PLIER and MAS5 for the Affymetrix Latin square data and our data of two sets of experiments using the same bone marrow but different types of microarrays and different assay. We found the computation for AUC of ROC at affycomp.biostat.jhsph.edu can be improved.