Materials and Methods
Animals: Collection and treatment
Periwinkles were purchased from a local seafood company and held for 3 weeks in the laboratory at 5*C in aerated full strength seawater (1000 mosmol). Control snails were sampled from this condition. Shells were quickly cracked open, foot and hepatopancreas were immediately removed, frozen in liquid nitrogen, and transferred to -80*C for storage until use. Anoxia treatment: Snails were transferred to closed jars with about 1-2 cm of seawater previously bubbled with 100% nitrogen gas for at least 10 min. Bubbling with nitrogen gas was continued for 15-20 min and then the jars were sealed and maintained at 5°C for of 1, 5, 12, 24, 48 and 144 hours. Freezing exposure: Animals were removed from the seawater, placed in closed plastic containers, and transferred to -8.0°C in an incubator for freezing. In initial tests, cooling and nucleation were monitored for a few snails by placing a thermistor inside the shell in contact with the mantle. Nucleation occurred within 45 minutes therefore, a 45 min cooling period was allowed before beginning to time the experimental freezing exposure times. After freezing/anoxia exposure, snails were rapidly removed and processed as above.
RNA preparation and cDNA library synthesis
Total RNA Total RNA was extracted from anoxic and frozen foot muscle using TrizolTM reagent (Gibco-BRL, Bethesda MD) according to manufaturers instructions. RNA quantity and purity was assessed with spectrophotometric methods and then stored at -70*.
mRNA isolation PolyA mRNA was isolated from total RNA using oligo dT cellulose (New England Biolabs) chromatography according to a protocol modified from Sambrook et al.. mRNA was quantified spectrophotometrically and stored at -70*C for future use.
cDNA library synthesis Libraries were constructed using a Uni-zap directional cloning kit according to Strategen Kit #066004 manufacturers instructions. Equal amounts of mRNA (totaling 5 *g) extracted from 1, 12 and 24 hour stressed foot muscle were combined for library synthesis. The anoxic library was made by Qin Yin Cai, the frozen library by Tamara English.
Differential screening of the cDNA library
Differential screening was carried out according to Cai and Storey (1996). Duplicate plaque lifts were prepared using Hybond N membranes (Amersham Life Sciences) and hybridized with 32P-labeled probe synthesized from mRNA. One lift is probed with stress probe, the other with control. Autoradiography films of the corresponding lifts are then compared. Plaques present on the stress film and not the control are putative stress-induced genes.
Putative clones were excised from the original NZY plate and added to 0.5 mL SM buffer and 100 *L chloroform. After removing cellular debris, the supernatant containing the phage was titred and plated onto separate 10 cm NZY plates to 50-100 pfu/mL. A new round of plaque lifts were made and screened with control and stress probe. This round of screening indicated the most likely candidates of freezing and anoxia induced genes, though another round of screening was required in some cases.
Phage deemed positive after screening were rescued in pBluescript by in vivo excision using the helper phage ExAssist according to Strategene instructions. The phage was then used to infect SOLR cells, from which, the phagemid containing the clone DNA was isolated using alkaline lysis, RNase treatment and phenol/chlorofom extraction.
Northern Blots: blotting protocol, and probing
Northern blots were used to assess changes of the levels of expression of putatively up-regulated clones over a time course of anoxia and freezing. Total RNA (16mg from each time point) was separated on a 1.5% formaldehyde denaturing agarose gel and blotting onto Nytran membrane (Scheiler and Scheull) according to a standard procedure (Sambrook et al.). When overnight transfer was complete, the RNA was fixed to the membrane with a UV cross-linker.
DNA inserts were isolated form putative up-regulated clones via digestion with EcoR1 and Xho1, the separated in a 1% agarose gel. Inserts were cut from the gel and purified using the Geneclean III kit (Bio101, Vista , CA). This DNA was used as the substrate for probe synthesis. Un-incorporated nucleotides were removed by centrifugation through a Sephadex G-50 column.
The Northern blots are pre-hybridized at 42*C for at least an hour in a formamide-based hybridization fluid. An amount of probe that provides 0.5 X 107 cpm per mL of hybe solution was boiled with salmon sperm DNA (10mg/mL) for 5 minutes added directly to the hybridization solution. Incubation was carried out overnight for 12-17 hours, depending on the cpm of the probe. The blot was removed from the tube and washed until the radioactivity was localized at a specific position and the rest of the blot showed only background and then exposed to autoradiography film for an appropriate time.
Transcript levels were quantified using Imagequant V3.22 to analyze the scans of the x-ray film. A Scanjet scanner with DeskScan (Hewlett Packard) was employed.
Gene se quencing was accomplished either manually using a USB Sequenase kit or by BioS&T in Lachine, Quebec
Computer software and internet databases The following web databases and computer software programs were used in this study:
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|English, T.E..; Storey, K.B..; (1998). Gene Up-regulation in Response to Anoxia or Freezing Stresses in the Marine Snail, Littorina littorea.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/|
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