Generation and Validation of a Shewanella oneidensis MR-1 Clone Set for Protein Expression and Phage Display

A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST- tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray) and other proteomic tools (e.g., mass spectrometry-based proteomic analysis), and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro

Characterization of sugar-insensitive mutants and analysis of sugar-regulated gene expression in Arabidopsis thaliana

Sugars serve as signaling molecules in plants, affecting gene expression and a number of developmental processes, but their precise role and the pathways through which they act are not well characterized. In addition, sugar-signaling pathways interact with the vast network of phytohormone-signaling pathways. In order to facilitate an understanding of sugar sensing and signaling pathways in Arabidopsis, sugar-insensitive mutants have been isolated for study and global genome analysis of wild-type and mutant responses to sugars and the phytohormones abscisic acid, ethylene, and gibberellin have been undertaken. Sugar-insensitive mutants were isolated based on their ability to form true leaves and expanded cotyledons in the presence of high concentrations (0.27 M to 0.34 M) of sucrose or glucose. The sis3 and sis6 mutants were chosen for further study. The sis3-1 mutation is recessive and is not linked to the presence of a T-DNA insert carried by the mutant. The sis3 mutation maps to the bottom arm of chromosome 3. The sis3 mutant is slightly insensitive to the effects of exogenously applied abscisic acid (ABA) on seed germination and root elongation but has a wild-type response to all other phytohormones tested. The sis6-1 mutant carries a cDNA encoding the At4g28240 putative wound-inducible gene on a T-DNA insert. The dominant nature of the mutation suggests that the five-fold overexpression of the cDNA in the mutant may be the cause of the sis phenotype. However, disruption of a gene by the T-DNA insertion is also a possible cause of the phenotype. The sis6 mutant is resistant to the inhibition of seed germination and root elongation caused by application of exogenous ABA. It is also resistant to the inhibition of germination caused by application of exogenous paclobutrazol (an inhibitor of gibberellin biosynthesis). This suggests a possible link between sugar and gibberellin signaling pathways. Analysis of gene expression in response to sugars and phytohormones was undertaken using the Affymetrix GeneChip Arabidopsis ATH 1 Genome Array which includes over 24,000 Arabidopsis genes. Both wild-type germinating seeds and adult plants were studied in order to begin determining the impact of the developmental age of the plant on sugar-regulated gene expression

Mathematical beliefs and achievement of pre-service primary teachers

Pre-service primary teachers in their first mathematics pedagogy subject at the University of Western Sydney completed three surveys: an achievement test of the mathematics they would be expected to teach; a survey of their beliefs about mathematics, mathematics teaching and mathematics learning; and a survey of their attitudes towards mathematics. this paper reports the data from the achievement test and the beliefs survey, and investigates the relationships between these

SUGAR-INSENSITIVE3, a RING E3 Ligase, Is a New Player in Plant Sugar Response1[OA]

Sugars, such as sucrose and glucose, have been implicated in the regulation of diverse developmental events in plants and other organisms. We isolated an Arabidopsis (Arabidopsis thaliana) mutant, sugar-insensitive3 (sis3), that is resistant to the inhibitory effects of high concentrations of exogenous glucose and sucrose on early seedling development. In contrast to wild-type plants, sis3 mutants develop green, expanded cotyledons and true leaves when sown on medium containing high concentrations (e.g. 270 mm) of sucrose. Unlike some other sugar response mutants, sis3 exhibits wild-type responses to the inhibitory effects of abscisic acid and paclobutrazol, a gibberellic acid biosynthesis inhibitor, on seed germination. Map-based cloning revealed that SIS3 encodes a RING finger protein. Complementation of the sis3-2 mutant with a genomic SIS3 clone restored sugar sensitivity of sis3-2, confirming the identity of the SIS3 gene. Biochemical analyses demonstrated that SIS3 is functional in an in vitro ubiquitination assay and that the RING motif is sufficient for its activity. Our results indicate that SIS3 encodes a ubiquitin E3 ligase that is a positive regulator of sugar signaling during early seedling development

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present