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| Status |
Public on Aug 01, 2013 |
| Title |
Global Analysis of de novo Transcription Following Biosynthetic Labeling in a Dinoflagellate, Karenia brevis |
| Organism |
Karenia brevis |
| Experiment type |
Expression profiling by array
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| Summary |
Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into role of de novo transcription in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed RNA in Karenia brevis. These isolated fractions were then used for analysis of global de novo transcription by hybridization to a K. brevis microarray. As previous microarray studies indicated that transcripts for pentatricopeptide repeat (PPR) proteins rapidly increased in response to nutrient addition, we queried the newly synthesized RNA pools at 1 and 4 h following nitrate addition to N-depleted cultures. Transcriptome-wide there was little evidence of changes in the rate of de novo transcription during the first 4 h, relative to that in N-depleted cells, and no evidence for increased PPR protein transcription. These results lend support to the growing consensus of post-transcriptional control of gene expression in dinoflagellates.
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| Overall design |
Cultures were acclimated to 10 µM nitrate for a minimum of two months during which time cultures were transferred weekly in log phase. For addition experiments, nutrient replete and N-limited 1 L cultures were grown to stationary phase (Day 9). Using sodium nitrate, 155 µM NO3 was added to stationary phase cultures. Cultures were exposed to 0.2 mM 4-thiouracil for 1 h to biosynthetically label newly transcribed RNA during the first hour post-N-addition (n=3) or during the fourth hour post-N-addition (n=3). N-limited cultures (n=3) were also exposed to 0.2 mM 4-thiouracil for 1 h to biosynthetically label newly transcribed RNA. Cultures were harvested after 1 h exposure to 4-thiouracil and total RNA extracted. Following extraction, RNA was biotinylated to allow for purification of the thiolated newly synthesized RNA from the total RNA pool using streptavidin coated magnetic beads. The newly transcribed RNA from each culture was independently labeled and hybridized to microarrys in a one color format. Based on the appearance of bioanlayzer profiles newly synthesized RNA was treated as mRNA in the labeling protocol.
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| Contributor(s) |
Morey JS, Van Dolah FM |
| Citation(s) |
23776661 |
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| Submission date |
Apr 18, 2013 |
| Last update date |
Nov 12, 2013 |
| Contact name |
Frances M Van Dolah |
| E-mail(s) |
[email protected]
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| Organization name |
Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Research
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| Street address |
219 Fort Johnson Rd
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| City |
Charleston |
| State/province |
SC |
| ZIP/Postal code |
29412 |
| Country |
USA |
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| Platforms (1) |
| GPL13364 |
NOAA Karenia brevis v3 (11K on 8x15K array format) |
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| Samples (9)
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| Relations |
| BioProject |
PRJNA197497 |
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