Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae

Yeast cells undergo diploid-specific developments such as spore formation via meiosis and pseudohyphal development under certain nutrient-limited conditions. Studies on these aspects require homozygous diploid mutants, which are generally constructed by crossing strains of opposite mating-type with the same genetic mutation. So far, there has been no direct way to generate and select diploids from haploid cells. Here, we developed a method for efficient construction of homozygous diploids using a PGAL1-HO gene (galactose-inducible mating-type switch) and a PSTE18-URA3 gene (counter selection marker for diploids). Diploids are generated by transient induction of the HO endonuclease, which is followed by mating of part of the haploid population. Since the STE18 promoter is repressed in diploids, diploids carrying PSTE18-URA3 can be selected on 5-fluoroorotic acid (5-FOA) plates where the uracil prototrophic haploids cannot grow. To demonstrate that this method is useful for genetic studies, we screened suppressor mutations of the complex colony morphology, strong agar invasion and/or hyper-filamentous growth caused by lack of the Hog1 MAPK in the diploid Σ1278b strain background. Following this approach, we identified 49 suppressor mutations. Those include well-known positive regulator genes for filamentous growth signaling pathways, genes involved in mitochondrial function, DNA damage checkpoint, chromatin remodeling, and cell cycle, and also previously uncharacterized genes. Our results indicate that combinatorial use of the PGAL1-HO and PSTE18-URA3 genes is suitable to efficiently construct and select diploids and that this approach is useful for genetic studies especially when combined with large-scale screening

Differential Hepatic Gene Expression Profile of Male Fathead Minnows Exposed to Daily Varying Dose of Environmental Contaminants Individually and in Mixture

Environmental contaminants are known to impair reproduction, metabolism and development in wild life and humans. To investigate the mechanisms underlying adverse effects of contaminants, fathead minnows were exposed to a number of endocrine disruptive chemicals (EDCs) including Nonylphenol (NP), bisphenol-A (BPA), Di(2-ethylhexyl) phthalate (DEHP), and a mixture of the three chemicals for 21 days, followed by determination of the liver transcriptome by expression microarrays. Pathway analysis revealed a distinct mode of action for the individual chemicals and their mixture. The results showed expression changes in over 980 genes in response to exposure to these EDC contaminants individually and in mixture. Ingenuity Pathway core and toxicity analysis were used to identify the biological processes, pathways and the top regulators affected by these compounds. A number of canonical pathways were significantly altered, including cell cycle & proliferation, lipid metabolism, inflammatory, innate immune response, stress response, and drug metabolism. We identified 18 genes that were expressed in all individual and mixed treatments. Relevant candidate genes identified from expression microarray data were verified using quantitative PCR. We were also able to identify specific genes affected by NP, BPA, and DEHP individually, but were also affected by exposure to the mixture of the contaminants. Overall the results of this study provide novel information on the adverse health impact of contaminants tested based on pathway analysis of transcriptome data. Furthermore, the results identify a number of new biomarkers that can potentially be used for screening environmental contaminants

Advanced Phase I/II Studies of Targeted Gene Delivery In Vivo: Intravenous Rexin-G for Gemcitabine-resistant Metastatic Pancreatic Cancer

Rexin-G, a nonreplicative pathology-targeted retroviral vector bearing a cytocidal cyclin G1 construct, was tested in a phase I/II study for gemcitabine-resistant pancreatic cancer. The patients received escalating doses of Rexin-G intravenously from 1 × 1011 colony-forming units (cfu) 2–3× a week (dose 0–1) to 2 × 1011 cfu 3× a week (dose 2) for 4 weeks. Treatment was continued if there was less than or equal to grade 1 toxicity. No dose-limiting toxicity (DLT) was observed, and no vector DNA integration, replication-competent retrovirus (RCR), or vector-neutralizing antibodies were noted. In nine evaluable patients, 3/3 patients had stable disease (SD) at dose 0–1. At dose 2, 1/6 patients had a partial response (PR) and 5/6 patients had SD. Median progression-free survival (PFS) was 3 months at dose 0–1, and >7.65 months at dose 2. Median overall survival (OS) was 4.3 months at dose 0–1, and 9.2 months at dose 2. One-year survival was 0% at dose 0–1 compared to 28.6% at dose 2, suggesting a dose–response relationship between OS and Rexin-G dosage. Taken together, these data indicate that (i) Rexin-G is safe and well tolerated, and (ii) Rexin-G may help control tumor growth, and may possibly prolong survival in gemcitabine-resistant pancreatic cancer, thus, earning US Food and Drug Administration's (FDA) fast-track designation as second-line treatment for pancreatic cancer

Co-optimization of SnS absorber and Zn(O,S) buffer materials for improved solar cells

Thin-film solar cells consisting of earth-abundant and non-toxic materials were made from pulsed chemical vapor deposition (pulsed-CVD) of SnS as the p-type absorber layer and atomic layer deposition (ALD) of Zn(O,S) as the n-type buffer layer. The effects of deposition temperature and annealing conditions of the SnS absorber layer were studied for solar cells with a structure of Mo/SnS/Zn(O,S)/ZnO/ITO. Solar cells were further optimized by varying the stoichiometry of Zn(O,S) and the annealing conditions of SnS. Post-deposition annealing in pure hydrogen sulfide improved crystallinity and increased the carrier mobility by one order of magnitude, and a power conversion efficiency up to 2.9% was achieved.United States. Department of Energy (Grant DE-EE0005329

Co-optimization of SnS absorber and Zn(O,S) buffer materials for improved solar cells

Thin-film solar cells consisting of earth-abundant and non-toxic materials were made from pulsed chemical vapor deposition (pulsed-CVD) of SnS as the p-type absorber layer and atomic layer deposition (ALD) of Zn(O,S) as the n-type buffer layer. The effects of deposition temperature and annealing conditions of the SnS absorber layer were studied for solar cells with a structure of Mo/SnS/Zn(O,S)/ZnO/ITO. Solar cells were further optimized by varying the stoichiometry of Zn(O,S) and the annealing conditions of SnS. Post-deposition annealing in pure hydrogen sulfide improved crystallinity and increased the carrier mobility by one order of magnitude, and a power conversion efficiency up to 2.9% was achieved.Engineering and Applied Science

Binding of lac repressor-GFP fusion protein to lac operator sites inserted in the tobacco chloroplast genome examined by chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) has been used to detect binding of DNA-binding proteins to sites in nuclear and mitochondrial genomes. Here, we describe a method for detecting protein-binding sites on chloroplast DNA, using modifications to the nuclear ChIP procedures. The method was developed using the lac operator (lacO)/lac repressor (LacI) system from Escherichia coli. The lacO sequences were integrated into a single site between the rbcL and accD genes in tobacco plastid DNA and homoplasmic transplastomic plants were crossed with transgenic tobacco plants expressing a nuclear-encoded plastid-targeted GFP-LacI fusion protein. In the progeny, the GFP-LacI fusion protein could be visualized in living tissues using confocal microscopy, and was found to co-localize with plastid nucleoids. Isolated chloroplasts from the lacO/GFP-LacI plants were lysed, treated with micrococcal nuclease to digest the DNA to fragments of ∼600 bp and incubated with antibodies to GFP and protein A-Sepharose. PCR analysis on DNA extracted from the immunoprecipitate demonstrated IPTG (isopropylthiogalactoside)-sensitive binding of GFP-LacI to lacO. Binding of GFP-LacI to endogenous sites in plastid DNA showing sequence similarity to lacO was also detected, but required reversible cross-linking with formaldehyde. This may provide a general method for the detection of binding sites on plastid DNA for specific proteins

Identification of a Bacterial Type III Effector Family with G Protein Mimicry Functions

SummaryMany bacterial pathogens use the type III secretion system to inject “effector” proteins into host cells. Here, we report the identification of a 24 member effector protein family found in pathogens including Salmonella, Shigella, and enteropathogenic E. coli. Members of this family subvert host cell function by mimicking the signaling properties of Ras-like GTPases. The effector IpgB2 stimulates cellular responses analogous to GTP-active RhoA, whereas IpgB1 and Map function as the active forms of Rac1 and Cdc42, respectively. These effectors do not bind guanine nucleotides or have sequences corresponding the conserved GTPase domain, suggesting that they are functional but not structural mimics. However, several of these effectors harbor intracellular targeting sequences that contribute to their signaling specificities. The activities of IpgB2, IpgB1, and Map are dependent on an invariant WxxxE motif found in numerous effectors leading to the speculation that they all function by a similar molecular mechanism

Improved Cu 2 O-Based Solar Cells Using Atomic Layer Deposition to Control the Cu Oxidation State at the p-n Junction

Chemistry and Chemical Biolog

Conservation of core gene expression in vertebrate tissues

Abstract Background Vertebrates share the same general body plan and organs, possess related sets of genes, and rely on similar physiological mechanisms, yet show great diversity in morphology, habitat and behavior. Alteration of gene regulation is thought to be a major mechanism in phenotypic variation and evolution, but relatively little is known about the broad patterns of conservation in gene expression in non-mammalian vertebrates. Results We measured expression of all known and predicted genes across twenty tissues in chicken, frog and pufferfish. By combining the results with human and mouse data and considering only ten common tissues, we have found evidence of conserved expression for more than a third of unique orthologous genes. We find that, on average, transcription factor gene expression is neither more nor less conserved than that of other genes. Strikingly, conservation of expression correlates poorly with the amount of conserved nonexonic sequence, even using a sequence alignment technique that accounts for non-collinearity in conserved elements. Many genes show conserved human/fish expression despite having almost no nonexonic conserved primary sequence. Conclusions There are clearly strong evolutionary constraints on tissue-specific gene expression. A major challenge will be to understand the precise mechanisms by which many gene expression patterns remain similar despite extensive cis-regulatory restructuring