Anthony D. Aragon

Characterization of Differentiated Quiescent and Nonquiescent Cells in Yeast Stationary-Phase Cultures

Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and nonquiescent (NQ) fractions before entering stationary phase. To understand this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity. Eleven mutants that affected one or both phenotypes in Q or NQ fractions were identified. NQ fractions exhibit a high level of petite colonies, and nine mutants affecting this phenotype were identified. Microarray analysis revealed >1300 mRNAs distinguished Q from NQ fractions. Q cell-specific mRNAs encode proteins involved in membrane maintenance, oxidative stress response, and signal transduction. NQ-cell mRNAs, consistent with apoptosis in these cells, encode proteins involved in Ty-element transposition and DNA recombination. More than 2000 protease-released mRNAs were identified only in Q cells, consistent with these cells being physiologically poised to respond to environmental changes. Our results indicate that Q and NQ cells differentiate significantly, with Q cells providing genomic stability and NQ cells providing nutrients to Q cells and a regular source of genetic diversity through mutation and transposition. These studies are relevant to chronological aging, cell cycle, and genome evolution, and they provide insight into complex responses that even simple organisms have to starvation.

Towards an understanding of the mechanism of action of praziquantel.

Although praziquantel (PZQ) has been used to treat schistosomiasis for over 20 years its mechanism of action remains unknown. We have developed an assay based on the transcriptional response of Schistosoma mansoni PR-1 to heat shock to confirm that while 6-week post-infection (p.i.) schistosomes are sensitive to PZQ, 4-week p.i. schistosomes are not. Further, we have used this assay to demonstrate that in mice this sensitivity develops between days 37 and 40 p.i. When PZQ is linked to the fluorophore BODIPY to aid microscopic visualization, it appears to enter the cells of intact 4 and 6-week p.i. schistosomes as well as mammalian NIH 3T3 cells with ease suggesting that the differential effects of PZQ is not based on cell exclusion. A transcriptomal analysis of gene expression between 4 and 6 weeks p.i. revealed 607 up-regulated candidate genes whose products are potential PZQ targets. A comparison of this gene list with that of genes expressed by PZQ sensitive miracidia reduced this target list to 247 genes, including a number involved in aerobic metabolism and cytosolic calcium regulation. Finally, we also report the effect of an in vitro sub-lethal exposure of PZQ on the transcriptome of S. mansoni PR-1. Annotation of genes differentially regulated by PZQ exposure suggests that schistosomes may undergo a transcriptomic response similar to that observed during oxidative stress.

Differential transcriptomic responses of Biomphalaria glabrata (Gastropoda, Mollusca) to bacteria and metazoan parasites, Schistosoma mansoni and Echinostoma paraensei (Digenea, Platyhelminthes)

A 70-mer-oligonucleotide-based microarray (1152 features) that emphasizes stress and immune responses factors was constructed to study transcriptomic responses of the snail Biomphalaria glabrata to different immune challenges. In addition to sequences with relevant putative ID and Gene Ontology (GO) annotation, the array features non-immune factors and unknown B. glabrata ESTs for functional gene discovery. The transcription profiles of B. glabrata (3 biological replicates, each a pool of 5 snails) were recorded at 12h post-wounding, exposure to Gram negative or Gram positive bacteria (Escherichia coli and Micrococcus luteus, respectively), or infection with compatible trematode parasites (Schistosoma mansoni or Echinostoma paraensei, 20 miracidia/snail), relative to controls, using universal reference RNA. The data were subjected to Significance Analysis for Microarrays (SAM), with a false positive rate (FPR) <or=10%. Wounding yielded a modest differential expression profile (27 up/21 down) with affected features mostly dissimilar from other treatments. Partially overlapping, yet distinct expression profiles were recorded from snails challenged with E. coli (83 up/20 down) or M. luteus (120 up/42 down), mostly showing up-regulation of defense and stress-related features. Significantly altered expression of selected immune features indicates that B. glabrata detects and responds differently to compatible trematodes. Echinostoma paraensei infection was associated mostly with down-regulation of many (immune-) transcripts (42 up/68 down), whereas S. mansoni exposure yielded a preponderance of up-regulated features (140 up/23 down), with only few known immune genes affected. These observations may reflect the divergent strategies developed by trematodes during their evolution as specialized pathogens of snails to negate host defense responses. Clearly, the immune defenses of B. glabrata distinguish and respond differently to various immune challenges.

Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

BackgroundAs carbon sources are exhausted, Saccharomyces cerevisiae cells exhibit reduced metabolic activity and cultures enter the stationary phase. We asked whether cells in stationary phase cultures respond to additional stress at the level of transcript abundance.ResultsMicroarrays were used to quantify changes in transcript abundance in cells from stationary phase cultures in response to stress. More than 800 mRNAs increased in abundance by one minute after oxidative stress. A significant number of these mRNAs encode proteins involved in stress responses. We tested whether mRNA increases were due to new transcription, rapid poly-adenylation of message (which would not be detected by microarrays), or potential release of mature mRNA present in the cell but resistant to extraction during RNA isolation. Examination of the response to oxidative stress in an RNA polymerase II mutant, rpb1-1, suggested that new transcription was not required. Quantitative RT-PCR analysis of a subset of these transcripts further suggested that the transcripts present in isolated total RNA from stationary phase cultures were polyadenylated. In contrast, over 2,000 transcripts increased after protease treatment of cell-free lysates from stationary phase but not exponentially growing cultures. Different subsets of transcripts were released by oxidative stress and temperature upshift, suggesting that mRNA release is stress-specific.ConclusionsCells in stationary phase cultures contain a large number of extraction-resistant mRNAs in a protease-labile, rapidly releasable form. The transcript release appears to be stress-specific. We hypothesize that these transcripts are associated with P-bodies.

Hyperspectral microarray scanning: impact on the accuracy and reliability of gene expression data

BackgroundCommercial microarray scanners and software cannot distinguish between spectrally overlapping emission sources, and hence cannot accurately identify or correct for emissions not originating from the labeled cDNA. We employed our hyperspectral microarray scanner coupled with multivariate data analysis algorithms that independently identify and quantitate emissions from all sources to investigate three artifacts that reduce the accuracy and reliability of microarray data: skew toward the green channel, dye separation, and variable background emissions.ResultsHere we demonstrate that several common microarray artifacts resulted from the presence of emission sources other than the labeled cDNA that can dramatically alter the accuracy and reliability of the array data. The microarrays utilized in this study were representative of a wide cross-section of the microarrays currently employed in genomic research. These findings reinforce the need for careful attention to detail to recognize and subsequently eliminate or quantify the presence of extraneous emissions in microarray images.ConclusionHyperspectral scanning together with multivariate analysis offers a unique and detailed understanding of the sources of microarray emissions after hybridization. This opportunity to simultaneously identify and quantitate contaminant and background emissions in microarrays markedly improves the reliability and accuracy of the data and permits a level of quality control of microarray emissions previously unachievable. Using these tools, we can not only quantify the extent and contribution of extraneous emission sources to the signal, but also determine the consequences of failing to account for them and gain the insight necessary to adjust preparation protocols to prevent such problems from occurring.

Multivariate curve resolution for hyperspectral image analysis: applications to microarray technology

Multivariate curve resolution (MCR) using constrained alternating least squares algorithms represents a powerful analysis capability for a quantitative analysis of hyperspectral image data. We will demonstrate the application of MCR using data from a new hyperspectral fluorescence imaging microarray scanner for monitoring gene expression in cells from thousands of genes on the array. The new scanner collects the entire fluorescence spectrum from each pixel of the scanned microarray. Application of MCR with nonnegativity and equality constraints reveals several sources of undesired fluorescence that emit in the same wavelength range as the reporter fluorphores. MCR analysis of the hyperspectral images confirms that one of the sources of fluorescence is due to contaminant fluorescence under the printed DNA spots that is spot localized. Thus, traditional background subtraction methods used with data collected from the current commercial microarray scanners will lead to errors in determining the relative expression of low-expressed genes. With the new scanner and MCR analysis, we generate relative concentration maps of the background, impurity, and fluroescent labels over the entire image. Since the concentration maps of the fluorescent labels are relativly uaffected by the presence of background and impurity emissions, the accuracy and useful dynamic range of the gene expression data are both greatly improved over those obtained by commercial microarray scanners.

Microarray based analysis of temperature and oxidative stress induced messenger RNA in Schistosoma mansoni

The bodys defense against schistosome infection can take many forms. For example, upon developing acute schistosomiasis, patients often have fever coinciding with larval maturation, migration and early oviposition. As the infection becomes established, the parasite comes under oxidative stress generated by the host immune system. The most common treatment for schistosomiasis is the anti-helminthic drug praziquantel. Its effectiveness, however, is limited due to its inability to kill schistosomes 2-4 weeks post-infection. Clearly there is a need for new anti-schistosomal drugs. We hypothesize that gene products expressed as part of a protective response against heat and/or oxidative stress are potential therapeutic targets for future drug development. Using a 12,166 element oligonucleotide microarray to characterize Schistosoma mansoni genes induced by heat and oxidative stress we found that 1878 S. mansoni elements were significantly induced by heat stress. These included previously reported heat-shock genes expressing homologs of HSP40, HSP70 and HSP86. One thousand and one elements were induced by oxidative stress including those expressing homologs of superoxide dismutase, glutathione peroxidase and aldehyde dehydrogenase. Seventy-two elements were common to both stressors and could potentially be exploited in the development of novel anti-schistosomal therapeutics.

Dual primer emulsion PCR for next- generation DNA sequencing.

We have developed a highly sensitive single-molecule clonal amplification method called dual primer emulsion PCR (DPePCR) for next-generation DNA sequencing. The approach is similar in concept to standard emulsion PCR; however, in DPePCR both primers are attached to the beads, therefore following PCR amplification, both strands of the PCR products are attached to the beads. The ends of each strand can be freed for analysis by restriction digestion of the bridged PCR fragments, which allows efficient paired-end sequencing of fragment libraries.

High-Quality Draft Genome Sequence of Actinobacterium Kibdelosporangium sp. MJ126-NF4, Producer of Type II Polyketide Azicemicins, Using Illumina and PacBio Technologies

ABSTRACT Here, we report the high-quality draft genome sequence of actinobacterium Kibdelosporangium sp. MJ126-NF4, producer of the type II polyketide azicemicins, obtained using Illumina and PacBio sequencing technologies. The 11.75-Mbp genome contains >11,000 genes and 22 polyketide and nonribosomal peptide natural product gene clusters.

Starvation-Associated Genome Restructuring Can Lead to Reproductive Isolation in Yeast

Knowledge of the mechanisms that lead to reproductive isolation is essential for understanding population structure and speciation. While several models have been advanced to explain post-mating reproductive isolation, experimental data supporting most are indirect. Laboratory investigations of this phenomenon are typically carried out under benign conditions, which result in low rates of genetic change unlikely to initiate reproductive isolation. Previously, we described an experimental system using the yeast Saccharomyces cerevisiae where starvation served as a proxy to any stress that decreases reproduction and/or survivorship. We showed that novel lineages with restructured genomes quickly emerged in starved populations, and that these survivors were more fit than their ancestors when re-starved. Here we show that certain yeast lineages that survive starvation have become reproductively isolated from their ancestor. We further demonstrate that reproductive isolation arises from genomic rearrangements, whose frequency in starving yeast is several orders of magnitude greater than an unstarved control. By contrast, the frequency of point mutations is less than 2-fold greater. In a particular case, we observe that a starved lineage becomes reproductively isolated as a direct result of the stress-related accumulation of a single chromosome. We recapitulate this result by demonstrating that introducing an extra copy of one or several chromosomes into naïve, i.e. unstarved, yeast significantly diminishes their fertility. This type of reproductive barrier, whether arising spontaneously or via genetic manipulation, can be removed by making a lineage euploid for the altered chromosomes. Our model provides direct genetic evidence that reproductive isolation can arise frequently in stressed populations via genome restructuring without the precondition of geographic isolation.