PIQA: pipeline for Illumina G1 genome analyzer data quality assessment.

SUMMARY: PIQA is a quality analysis pipeline designed to examine genomic reads produced by Next Generation Sequencing technology (Illumina G1 Genome Analyzer). A short statistical summary, as well as tile-by-tile and cycle-by-cycle graphical representation of clusters density, quality scores and nucleotide frequencies allow easy identification of various technical problems including defective tiles, mistakes in sample/library preparations and abnormalities in the frequencies of appearance of sequenced genomic reads. PIQA is written in the R statistical programming language and is compatible with bustard, fastq and scarf Illumina G1 Genome Analyzer data formats. AVAILABILITY: The PIQA pipeline, installation instructions and examples are available at the supplementary web site (http://bioinfo.uh.edu/PIQA).

Accelerated photobleaching of a cyanine dye in the presence of a ternary target DNA, PNA probe, dye catalytic complex: a molecular diagnostic.

In many settings, molecular testing is needed but unavailable due to complexity and cost. Simple, rapid, and specific DNA detection technologies would provide important alternatives to existing detection methods. Here we report a novel, rapid nucleic acid detection method based on the accelerated photobleaching of the light-sensitive cyanine dye, 3,3'-diethylthiacarbocyanine iodide (DiSC(2)(3) I(-)), in the presence of a target genomic DNA and a complementary peptide nucleic acid (PNA) probe. On the basis of the UV-vis, circular dichroism, and fluorescence spectra of DiSC(2)(3) with PNA-DNA oligomer duplexes and on characterization of a product of photolysis of DiSC(2)(3) I(-), a possible reaction mechanism is proposed. We propose that (1) a novel complex forms between dye, PNA, and DNA, (2) this complex functions as a photosensitizer producing (1)O(2), and (3) the (1)O(2) produced promotes photobleaching of dye molecules in the mixture. Similar cyanine dyes (DiSC(3)(3), DiSC(4)(3), DiSC(5)(3), and DiSC(py)(3)) interact with preformed PNA-DNA oligomer duplexes but do not demonstrate an equivalent accelerated photobleaching effect in the presence of PNA and target genomic DNA. The feasibility of developing molecular diagnostic assays based on the accelerated photobleaching (the smartDNA assay) that results from the novel complex formed between DiSC(2)(3) and PNA-DNA is under way.

A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination.

With current plant transformation methods ( Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable. An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombination (SSR) systems, such as Cre/ lox. Cre has been shown in numerous instances to mediate lox site-specific recombination in animal and plant cells. By incorporating the Cre/ lox SSR system into a bacterial artificial chromosome (BAC) vector, a more precise evaluation of large DNA inserts for genetic complementation should be possible. Site-specific insertion of DNA into predefined sites in the genome may eliminate unwanted 'position effects' caused by the random integration of exogenously introduced DNA. In an effort to make the Cre/ lox system an effective tool for site-directed integration of large DNAs, we constructed and tested a new vector potentially capable of integrating large DNA inserts into plant and fungal genomes. In this study, we present the construction of a new BAC vector, pBACwich, for the system and the use of this vector to demonstrate SSR of large DNA inserts (up to 230 kb) into plant and fungal genomes.

T-2 toxin inhibits mitochondrial function in yeast.

T-2 toxin inhibits oxygen consumption of whole cells and purified mitochondria of Saccharomyces cerevisiae. Inhibition of mitochondrial respiration is not relieved by 2, 4-dinitrophenol, indicating that T-2 toxin inhibits mitochondrial function at the level of the electron transport chain. T-2 toxin inhibition of state 3 respiration (with succinate) is overcome by N, N, N', N'-tetramethyl-p-phenylenediamine, indicating inhibition of site II of the electron transport chain. T-2 toxin inhibits mitochondrial succinate dehydrogenase activity and increases mitochondrial NADH dehydrogenase activity.

Trichothecene synergism, additivity, and antagonism: the significance of the maximally quiescent ratio.

The interactive effect of the combinations of trichothecene mycotoxins often found in fungus infected plants, contaminated grain, and other biological systems is poorly understood. Growth inhibition of the yeast Kluyveromyces marxianus was used to measure the effects of HT-2 toxin, roridin A, and T-2 toxin as individual toxins or as binary mixtures. A value, the combination index, was derived which indicates the interactive effects of a binary mixture of toxins. The interaction is affected by the ratio of the individual toxins, and the percent inhibition of yeast growth. Generally the interaction of T-2 toxin and roridin A or T-2 toxin and HT-2 toxin changes from antagonistic when they cause a low percent inhibition of yeast growth to synergistic when they cause a high percent inhibition of yeast growth. Additionally, any two trichothecenes have a unique ratio, which we name the maximally quiescent ratio (or MQR), where there is the least change in the type and intensity of their interaction. The maximally quiescent ratio in this case has helped to define the nature of toxin interactions and could be used to provide insights into hormone, immune system, developmental, enzyme, and gene regulation, combined drug therapy, and the action of mixtures of natural or synthetic toxins, carcinogens, pesticides, and environmental pollutants.

Cre-lox site-specific recombination between Arabidopsis and tobacco chromosomes.

To create hybrid chromosomes, we tested the Cre-lox system to mediate recombination between Arabidopsis thaliana and Nicotiana tabacum chromosomes. Protoplasts of the two plants were fused to allow site-specific recombination to join a promoter from tobacco to a hygromycin resistance coding-region from Arabidopsis. The expected recombination junction was detected in hygromycin-resistant calli. Analysis of one hybrid suspension cell line revealed the presence of markers corresponding to the north arm of Arabidopsis chromosome III, but not markers from other chromosome arms. However, these markers were not detected in regenerated plants. With a second hybrid cell line we obtained a single hygromycin-resistant progeny from approximately 18 000 self-fertilized seeds of one regenerated plant. Molecular analysis of this hybrid indicated that a small portion of the north arm of Arabidopsis chromosome V is present in the tobacco genome. However, neither the recombination junction nor Arabidopsis DNA was detected in tissue from the plant grown without selection or in the subsequent generation. Thus interspecies transfer of a chromosome arm between plant cells is possible, but maintenance of the hybrid chromosome in a plant is unlikely. The feasibility of site-specific recombination between genomes of different species offers new possibilities for engineering hybrid chromosomes that may be maintained in cell culture.

Effects of growth temperature on toxicity of T-2 toxin and roridin A to yeast.

In yeasts, growth temperature is known to affect the membrane phospholipid content. The effect of temperature on the growth inhibition of Kluyveromyces marxianus and Saccharomyces cerevisiae by the trichothecene mycotoxins, T-2 toxin and roridin A, was investigated. Examination of EC50 values for T-2 toxin and roridin A showed that these toxins were least inhibitory to both yeasts at 30 and 25 degrees C, respectively. Increasing or decreasing growth temperature from these temperatures gradually increased the inhibitory effect of the trichothecene mycotoxins. Temperature may affect the toxicity of the trichothecenes to the yeasts by regulating the composition of yeast cell membranes.

Effects of T-2 toxin on ethanol production by Saccharomyces cerevisiae.

A trichothecene mycotoxin, T-2 toxin, inhibits several aspects of cellular physiology in Saccharomyces cerevisiae, including protein synthesis and mitochondrial functions. We have studied growth of, glucose utilization by, and ethanol production by S. cerevisiae and show that they are inhibited by T-2 toxin between 20 and 200 micrograms/ml in a dose-dependent manner. At 200 micrograms/ml, T-2 toxin causes cell death. This apparent inhibition of ethanol production was found to be the result of growth inhibition. On the basis of biomass or glucose consumption, T-2 toxin increased the amount of ethanol present in the culture. This suggests that T-2 inhibits oxidative but not fermentative energy metabolism by inhibiting mitochondrial function and shifting glucose catabolism toward ethanol formation. As T-2 toxin does not directly inhibit ethanol production by S. cerevisiae, this system could be used for ethanol production from trichothecene-contaminated grain products.

The occurrence of inducible anti-Escherichia coli activity in hemolymph from the migratory grasshopper, Melanoplus sanguinipes.

1. Inducible antibacterial activity against an Escherichia coli strain has been shown to occur in the hemolymph of Melanoplus sanguinipes. 2. This anti-E. coli activity was induced by the infection with Serratia marcescens or E. coli K12 but not Enterobacter cloacae. 3. The greatest anti-E. coli activity was observed in hemolymph extracted from grasshoppers 6 hr after the injection of any of the two species of bacteria. 4. Anti-E. coli activity disappeared in hemolymph extracted from grasshoppers 8 hr after injection or longer.

Effect of T-2 toxin and verrucarin A in combination on Kluyveromyces marxianus.

The growth inhibitory effects of combinations of T-2 toxin and verrucarin A on the yeast Kluyveromyces marxianus was studied. A combination index value was derived to indicate the type of interaction that existed between the binary mixture of these two toxins at various ratios and the target yeast cells. The type of interaction was dependent on the ratio of the toxins used to attain a particular level of growth inhibition. Further, the least change in the type and intensity of interaction or the maximally quiescent ratio (MQR) was found to be unique to the growth medium. In a rich medium, the MQR was 1.0 microgram/ml T-2 toxin:0.75 microgram/ml verrucarin A, where the two toxins had a very stable synergistic interaction over a 2 or 3 log value concentration range. Decreasing the nutrients changed the MQR to 1.0 micrograms/ml T-2 toxin:0.38 microgram/ml verrucarin A. Halving the concentration of the cells in the assay changed the MQR to 1.0 microgram/ml T-2 toxin:6.0 micrograms/ml verrucarin A. We have previously shown that the hierarchy of trichothecene toxicity in yeast bioassay is verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin. The MQR of these toxins in combination with T-2 toxin follows the same order. This study shows an exception to the above order in that verrucarin A and roridin A exchange places.

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to T-2 toxin.

T-2 toxin, a trichothecene mycotoxin, inhibits the growth of Saccharomyces cerevisiae. We have isolated nine spontaneous S. cerevisiae mutants resistant to this toxin. The mutants were distinguished from the wild type according to their degree of resistance to T-2 toxin on media with dextrose or glycerol as the carbon source. Generation time, mutation stability and level of cross-resistance to roridin A, another trichothecene, were determined for each mutant. The T-2 toxin resistant mutants were further characterized by subsequent tests involving cross-resistance and collateral sensitivity to chlorampenicol, neomycin, paromomycin, ethidium bromide and thiolutin. Mutants have been placed into three subgroups and the mechanism of T-2 toxin resistance in each group has been postulated. Mutant HK1 is the first S. cerevisiae isolate resistant to roridin A. One particular isolate, mutant HK11, carries a single recessive nuclear mutation. This mutation was termed ttt (for T-2 toxin resistant).