Project description:Pseudo-nitzschia multiseries (Ps-n) is a toxigenic marine diatom that produces the neurotoxin, domoic acid. We screened for candidate genes that may be involved in domoic acid production by determining changes in transcript profiles in Ps-n cultures that were in late exponential (low-domoic-acid-producing) vs. stationary (high-domoic-acid-producing) growth states. We also identified a number of candidate reference genes for future RT-qPCR studies, based on their stability in this study.
Project description:DNA oligonucleotide microarrays were designed with 307 probes for 96 internal transcribed spacer (ITS1, located between 18S and 26S rRNA genes) sequences of known species and strains from the genus Pseudo-nitzschia (Bacillariophyceae). In addition, microarrays also carried 1893 probes targeting ITS1 aequences of marine Crenarchaeota and Alphaproteobacteria of SAR11 clade. In order to assign microarray profiles to Pseudo-nitzschia ribotypes and species and to 'train' the data analysis system, we grew cultures of Pseudo-nitzschia in the laboratory with identities confirmed through rDNA sequence analysis. In total, 9 cultures and 35 environmental water samples were hybridized to microarrays, in some cases, in duplicate or triplicate. Analysis of microarray data allowed us to identify and map Pseudo-nitzschia spp. in the coastal waters along Washington and Oregon coast of the Eastern Pacific Ocean, and to observe seasonal changes in diatom community composition.
Project description:DNA oligonucleotide microarrays were designed with 307 probes for 96 internal transcribed spacer (ITS1, located between 18S and 26S rRNA genes) sequences of known species and strains from the genus Pseudo-nitzschia (Bacillariophyceae). In addition, microarrays also carried 1893 probes targeting ITS1 aequences of marine Crenarchaeota and Alphaproteobacteria of SAR11 clade. In order to assign microarray profiles to Pseudo-nitzschia ribotypes and species and to 'train' the data analysis system, we grew cultures of Pseudo-nitzschia in the laboratory with identities confirmed through rDNA sequence analysis. In total, 9 cultures and 35 environmental water samples were hybridized to microarrays, in some cases, in duplicate or triplicate. Analysis of microarray data allowed us to identify and map Pseudo-nitzschia spp. in the coastal waters along Washington and Oregon coast of the Eastern Pacific Ocean, and to observe seasonal changes in diatom community composition. Total DNA was isolated from 9 Pseudo-nitzschia laboratory cultures and 35 environmental water samples collected during 7 field campaigns in 2007-2009. The environmental samples were collected at distances of 5 to 55 km from the coast, along the following transects in the Pacific Ocean covering over 300 km of the coastline: La Push (LP), Grays Harbor (GH), Columbia River (CR), and Newport Hydroline (NH). The DNA samples were subjected to PCR amplification with the primers specific for ITS1 sequences. The resultant biotin-labeled target samples were analyzed using microarray hybridization with the CombiMatrix ElectraSense 4X2K format. Out of 44 analyzed samples, 40, 2, and 2 were used for single, duplicate and triplicate hybridizations, respectively.
Project description:Pseudo-nitzschia multiseries (Ps-n) is a toxigenic marine diatom that produces the neurotoxin, domoic acid. We screened for candidate genes that may be involved in domoic acid production by determining changes in transcript profiles in Ps-n cultures that were in late exponential (low-domoic-acid-producing) vs. stationary (high-domoic-acid-producing) growth states. We also identified a number of candidate reference genes for future RT-qPCR studies, based on their stability in this study. Ps-n RNA was extracted from late exponential and stationary phase cultures. Experiments included one axenic culture, and two non-axenic cultures. Experiments were dye-swapped to account for differences in dye labeling and detection efficiencies: a) the axenic culture experiment included 4 technical replicate arrays (i.e., 2 dye-swapped experiments = 4 total hybridizations), and b) the non-axenic culture experiments each included 6 technical replicate arrays (i.e., 3 dye-swapped experiments = 6 total hybridizations).
Project description:Cultures of two Pseudo-nitzschia multistriata strains of opposite mating type were placed in a bipartite glass apparatus with a membrane separating them. This allows perception of sexual cues. Samples were taken at 2 hours and 6 hours from the start of the experiment and gene expression was compared to the same strains grown in monocultures.
