ABSTRACT: Salvia is an important genus from the Lamiaceae with approximately 1000 species distributed globally. Several Salvia species are commercially important because of their medicinal and culinary properties. We report the construction of the first fingerprinting array for Salvia species enriched with polymorphic and divergent DNA sequences and demonstrate the potential of this array for fingerprinting several economically important members of this genus. In order to generate the Salvia Subtracted Diversity Array (SDA), a Suppression Subtractive Hybridization (SSH) was performed between a pool of ten Salvia species and a pool of non-angiosperm and angiosperms (excluding the Lamiaceae) to selectively isolate Salvia-specific sequences. A total of 285 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. DNA fingerprints were obtained for fifteen Salvia genotypes including three that were not part of the original subtraction pool. Hierarchical cluster analysis indicated that the Salvia-specific SDA was capable of differentiating closely related species of S. officinalis and S. miltiorrhiza and was also able to reveal genetic relationships consistent with geographical origins. Species-specific features were also found for S. elegans, S. officinalis, S. sclarea, S. przewalskii and S. runcinata.
Project description:Echinacea, native to the Canadian prairies and the prairie states of the United States, has a long tradition as a folk medicine for the Native Americans. Currently, Echinacea are among the top 10 selling herbal medicines in the U.S. and Europe, due to increasing popularity for the treatment of common cold and ability to stimulate the immune system. However, the genetic relationships within the species of this genus are unclear, which makes difficult the authentication of the species used for the medicinal industry. We report the construction of a novel Subtracted Diversity Array (SDA) for Echinacea species and demonstrate the potential of this array for isolating highly polymorphic sequences. In order to selectively isolate Echinacea-specific sequences, a Suppression Subtractive Hybridization (SSH) was performed between a pool of twenty-four Echinacea genotypes and a pool of other angiosperms and non-angiosperms. A total of 283 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. Twenty-seven Echinacea genotypes, including four that were not used in the array construction, could be successfully discriminated. Interestingly, unknown samples of E. paradoxa and E. purpurea could be unambiguously identified from the cluster analysis. Furthermore, this Echinacea-specific SDA was also able to isolate highly polymorphic sequences. Five out of the eleven most discriminatory features matched to known retrotransposons.
Project description:An anthropogenic chemical contaminant commonly identified in aquatic receiving environments is the non-steroidal anti-inflammatory drug (NSAID), ibuprofen(IBF). While the role of ibuprofen in target organisms is known, there exists a paucity of data on the impact of exposure to non-target wildlife species. In the case of frog species, normal development and environmental fitness involves the actions of the thyroid hormones (THs), particularly at key points in the life cycle. We investigated whether exposure of premetamorphic North American bullfrog (Rana catesbeiana) tadpoles to IBF altered their response to treatment with an exogenous dose of thyroid hormone (T3). Six animals randomly selected from each treatment were examined for the status of the hepatic transcriptome using MAGEX DNA array analysis.
Project description:Photosynthesis is arguably the most important biological process on earth. In plants, energy harvested in photosynthesis is converted into sugar and starch, which are important products from species with agronomic interest. During the photosynthesis in the chloroplast, the intermediate carbon metabolites (triose phosphates) produced by the Calvin cycle can either be exported to the cytosol for sucrose synthesis or stay in the chloroplast for starch synthesis (formation). Two fructose-1,6-bisphosphatase (FBPase) enzymes, the chloroplastidial (cpFBPaseI) and the cytosolic (cyFBPase) isoforms, catalyse the first irreversible step during the conversion of triose phosphate to starch or sucrose, respectively. Recently, another cpFBPase isoform (cpFBPaseII) with unknown function was identified. It has been reported that the reduction of cyFBPase or cpFBPaseI activity leads to an alteration in starch and sucrose content. In our laboratory, Arabidopsis thaliana knock-out mutants for the cyFBPase and cpFBPaseI are now available. The objective this research project is to identify and functionally characterize genes differentially expressed in Arabidopsis thaliana knock-out mutants lacking FBPase genes. We make use of high throughput methodologies, such as the transcriptomic and proteomic analyses which represent invaluable tools to identify new loci responsible for agronomically important traits. Three independent biological replicates were used for each type of sample. Three hybridizations were performed, representing the three independent biological replicates, being one of them a dye swap
Project description:A screening process was used to determine the aciduric capacities of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at pH 5.0 in the presence 4 different organic acids. Food and clinical isolates tended to be more resistant but strain genetic groupings were found to be only weakly linked to sodium diacetate (SDA)-resistance. Representative and comparatively aciduric food isolates FW04/0023 and FW04/0025 were found to accumulate reduced levels of acetate anion and K+ ion during growth in the presence of SDA, compared to more acid sensitive reference strains EGD (ATCC BAA-739) and ATCC 19111. The aciduric nature of FW04/0023 and FW04/0025 was also reflected by their comparatively high tolerance to pH 2.4 acid challenge; a property boosted by the presence of SDA, and growth-phase dependent acid tolerance. Transcriptomic analyses revealed increased levels of SDA did not activate or promulgate the response of any of the known acid protective mechanisms, except for acetoin biosynthesis. Elevated levels of unprotonated SDA (20 mM SDA at pH 5.0) was found to have broad effects on gene expression that could be differentiated from effects caused by mildly acidic conditions (pH 5.0) and substantial strain variation was also found. Collated SDA mainly effect genes associated with virulence, cell wall processes, DNA repair, and cofactor and lipid biosynthesis. Strain FW04/0025 was more responsive to elevated unprotonated SDA increasing the expression of 222 genes (>2-fold change, p<0.05), compared to 112 genes for strain EGD. Key differences between the strains in relation to SDA-enhanced transcript abundance in FW04/0023 were primarily associated with the cell wall, oxidative stress management, intermediary metabolism, and several hypothetical proteins. This complement of different responses could potentially alter phenotypes resulting in different cell envelope properties and metabolic properties that effect accumulation of acetate anion. The data demonstrates that amongst different L. monocytogenes strains acetate has differing effects that may ultimately influence growth efficiency under stressful conditions relevant to acidic foods and the gastrointestinal environment. The microarray component of the experiments had the aim of determining: i) To examine the affect of elevated levels of unprotonated sodium diacetate (21 mM sodium diacetate added to cultures at pH 5.0, resulting in ~7.7 mM unprotonated acetate) compared to mild acid stress at pH 5.0 in which lower levels of acetate accumulates through natural end-product formation. ii) To examine the gene expression responses of two L. monocytogenes strains that had different intrinsic acid resistances, including an organic acid resistant strain FW04/0025 and a more sensitive reference strain EGD on which genome the microarray oligonucleotide set is based. Two to four biologically replicated control cultures were labelled with Cy3 for each treatment sample with two sets of experimental conditions investigated for two different L. monocytogenes strains . Condition 1) â Mildly acidic stress: Control culture (Cy3-labelled RNA) - strains were grown to early- mid exponential growth phase at 25°C (in a shaking water bath) in brain-heart infusion broth at pH 7.3. Test cultures (Cy5-labelled RNA) â strains grown to early- mid exponential growth at 25°C phase in BHI broth adjusted to pH 5.0 (in a shaking water bath). Condition 2) â Organic acid (sodium diacetate) stress: Control culture (Cy3-labelled RNA) - strains were grown to exponential growth phase at 25°C (in a shaking water bath) in BHI broth at pH 5.0. Test cultures (Cy5-labelled RNA) â strains grown to exponential growth at 25°C phase in BHI broth adjusted to pH 5.0 and amended with 21 mM (0.3% w/v)sodium diacetate (in a shaking water bath).
Project description:Long-term exposure of Atlantic salmon to 19M-BM-0C resulted in cardiac gene and protein expression changes indicating that the unfolded protein response, vascularization, remodeling of connective tissue and altered innate immune responses were part of the cardiac acclimation or response to elevated temperature Heart tissue from Atlantic salmon (RNA poooled from N=3 fish per 3 replicate tanks) reared under normal (14 deg C) and elevated (19 deg C) temperatures were sampled after 21 (short-term) and 56 days (long-term acclimation), and gene expression changes between the elevated and the control temperature were assessed using microarray analysis.
Project description:Microsporidia have attracted much attention because they infect a variety of species ranging from protists to mammals, including immunocompromised patients with AIDS or cancer. Aside from the study on Nosema ceranae, few works have focused on elucidating the mechanism in host response to microsporidia infection. Nosema bombycis is a pathogen of silkworm pM-CM-)brine that causes great economic losses to the silkworm industry. Detailed understanding of the host (Bombyx mori) response to infection by N. bombycis is helpful for prevention of this disease. The 23 K silkworm genome array was used to investigate host responses (i.e., Bombyx mori) occurring at 2, 4, 6 and 8 d post-infection by Nosema bombycis.We focused on elucidating the mechanism of the host response to microsporidia infection, especially for the investigation of host immune response . The third instar molted silkworm larvae were in oral infected by Nosema bombycis. In order to known the silkworm host response to Nosema bombycis infection at different time points, samples of infected larvae (i.e., the treatment set) and uninfected larvae (i.e., the control set) were collected at 2, 4, 6 and 8 dpi for RNA extraction and array hybridization. The obtained data were usd to investigate on the interplay of the genome-wide expression profile of hosts.
Project description:DNA microarrays were used to profile gene expression in normal murine splenic cells vs. prion infected splenic cells with the aim of identifying host factors involved in prion propagation and/or useful as biomarkers for early diagnosis. Two condition experiment, prion infected murine splenic cells vs. normal murine splenic cells: 4 infected biological replicates, 2 control biological replicates, 2 technical replicates (dye-swaps) for each competitive hybridization. Samples were run on 4 different arrays: MPB Murine 16K BMAP-MSV printrun 7.1 microarray MPB Murine 16K BMAP-MSV printrun 9.1 microarray MPB Murine 32K Oligo printrun 4.1 microarray Agilent 4x44K Whole Mouse Genome
Project description:WD40 motif-containing Msi1-like (MSIL) proteins play pleiotropic cellular functions as a negative regulator of the Ras/cAMP-pathways and a component of chromatin assembly factor-I (CAF-I), and yet have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans, which can cause fatal meningoencephalitis in humans. Notably, Msl1 was not a functional ortholog for the yeast Msi1 but played pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners but mainly Ras-independently. Msl1 negatively controlled antioxidant melanin production and sexual differentiation, which can be repressed by inhibiting the cAMP-signaling pathways. In contrast, Msl1 controlled thermotolerance, diverse stress responses, and antifungal drugs resistance in Ras/cAMP-independent manners. Cac2, which is the second CAF-I component, appeared to play both redundant and distinct function with Msl1. Msl1 is required for full virulence of C. neoformans. Transcriptome and proteomic analysis identified a group of Msl1-regulated genes or -interacting proteins, respectively, which mostly include stress-related genes, including HSP12, HSP78, SSA1, SSA4, and STM1. Furthermore, we identified the third putative component of CAF-1, Rlf2, in C. neoformans. In conclusion, this study demonstrated the pleiotropic roles of Msl1 in human fungal pathogen C. neoformans, providing a novel antifungal therapeutic target. There is more than 95% genome homology between JEC21 and H99. Therefore, 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligos are used in this analysis. Total RNAs are extracted from 2 strains from H99 (H99 wild-type strain (Cryptococcus neoformans var. grubii serotype A), msl1M-NM-^T). 3 biological replicate experiments are performed for each strain. We use the mix of all total RNAs from this experiment as the control RNA. We use Cy3 as the test sample dye and Cy5 as the control dye.
Project description:The high osmolarity glycerol response (HOG) pathway plays a pivotal role in the stress response, virulence regulation, and differentiation of fungi, including Cryptococcus neoformans that causes fatal meningoencephalitis. Core signaling components of in the HOG pathway, including the Tco-Ypd1-Ssk1 phosphorelay system and the Ssk2-Pbs2-Hog1 MAPK module, have been elucidated but its downstream transcription factors remain unclear. Here we demonstrated that Atf1 with a basic leucine zipper domain is the transcription factor downstream of Hog1 in C. neoformans. We found that ATF1 expression was differentially regulated by oxidative damaging agents, mainly in a Hog1-dependent, but Mpk1-independent, manner. Interestingly, Atf1 not only promoted oxidative stress response and adaptation, but also played an opposing role to Hog1 in the process. Atf1 primarily localized to the nucleus under both unstressed and oxidative stress conditions in a Hog1-independent manner. Our data demonstrated that Atf1 promoted pheromone production and sexual differentiation under negative control by Hog1. Finally, a DNA microarray-based transcriptome analysis of the atf1M-bM-^HM-^F mutant under unstressed and oxidative stress conditions revealed that Atf1 regulated oxidative stress response genes, including a sulfiredoxin gene (SRX1). Intriguing, the array data further demonstrated that Atf1 modulated basal expression of genes involved in DNA repair and genotoxic stress response. Supporting this, we found that the atf1M-bM-^HM-^F mutant was highly sensitive to genotoxic agents. In conclusion, this study provided a further insight into the Hog1-dependent oxidative and genotoxic stress response and differentiation mechanism of C. neoformans. There are more than 95% of genome homology between JEC21 and H99. Therefore 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted 2 conditions (with or without treatment of Hydroten peroxide) from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), and atf1M-NM-^T). We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy3 as Sample dye and Cy5 as a control dye.
Project description:Heritable variation in gene expression has been largely studied in model organisms by taking advantage of pure and inbred lines. Comparative studies on organisms with contrasting evolutionary history, population size and genome architecture have been hindered by practical considerations, as many of these species are poorly suited for breeding. We have developed a method for determining expression variation in an undomesticated tree species by directly measuring the segregation of gene expression in the haploid meiotic products of a single diploid individual. We show the utility of our approach by identifying expression variation that segregates consistent with one causal variant and identify an unexpectedly large number of these Mendelian expression traits. The megagametophyte approach opens the doors for the application of genetical genomics approaches in a large number of undomesticated plant species. Transcriptomics data from 18 megagametophytes of two unrelated mother trees. In tree 80112, all of the 18 megagametophytes were analyzed on to different microarrays with two different dyes. In tree 77111, 16 megagametophytes were replicated as above.