Identification of an intronic enhancer in the tilapia glutamine synthetase gene.
In preparation (JBC)
- Organism: Oreochromis mossambicus
- Instrument: impact II
Ecological Proteomics, Euryhaline fish, DIA assay, Tilapia cell line
- Lab head:
A reporter assay was used in the tilapia OmB cell line for identifying osmolality/ salinity responsive enhancers in the Oreochromis mossambicus glutamine synthetase gene. Genomic DNA comprising regulatory regions of this gene was isolated and analyzed using dual luciferase enhancer trap reporter assays. Using targeted proteomics, hyperosmotic induction of this tilapia glutamine synthetase gene was shown to be significant (four-fold) at the protein level. The hyperosmotic upregulation of glutamine synthetase was completely inhibited in the presence of actinomycin D. Therefore, the mechanism of hyperosmotic glutamine synthetase induction is transcriptional. This study identifies intron 1 of the glutamine synthetase gene as a major target of hyperosmotically induced transcription. We show that intron 1 contains an OSRE1 cis-regulatory element that requires intron mediated enhancement (IME) to confer hyperomosmotic induction of the tilapia glutamine synthetase gene.
Tryptic peptides from each sample (200 ng on column) were injected with a nanoAcquity sample manager (Waters), trapped for 1 min at 15 µL/min on a Symmetry trap column (Waters 186003514), and separated on a 1.7µm particle size BEH C18 column (250mm x 75µm, Waters) by reversed phase LC using a nanoAcquity binary solvent manager (Waters). A 125 min linear gradient ranging from 3% to 35% acetonitrile was used. Peptides were ionized by nano-ESI using a pico-emitter tip (New Objective) and analyzed by an Impact II UHR-QTOF mass spectrometer (Bruker). Batch-processing of samples was controlled with Hystar (Bruker). The mass range was set to 490 – 910 m/z at 15 Hz scan rate with an isolation width of 11 m/z (1 m/z overlap). Spectral libraries were created from prior precursor-dependent MS1 data (< 20 ppm mass error). DIA data for targeted proteins were statistically analyzed using Skyline 4 (MacCossLab, UW).
Tilapia OmB cells were dosed for 24 h in either hyperosmotic (650 mosm/kg) or isosmotic (315 mosm/kg control) media. Two additional treatment groups were exposed to 10 µM actinomycin D during dosing for 24 h in either hyperosmotic (650 mosm/kg) or isosmotic (315 mosm/kg control) media. Replicates represent different cell culture dishes from which proteins were extracted and processed independently.
Created on 10/31/19, 10:48 AM