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Development of Biomarkers from seagrass Posidonia oceanica
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Technical Annex (Chapter 19) Development of Biomarkers from seagrass Posidonia oceanica A. Schoendorf, I. Benveniste, J.P. Salaiin Institut de Biologie Mol6culaire des Plantes, CNRS-UPR 406, 28 rue Goethe, 67083 Strasbourg Cedex, France
Material White (non chlorophyllous) and green (chlorophyllous) parts of Posidonia oceanica leaves (100g fresh weight) were collected from polluted area of the French Riviera (Nice, Monaco, La Foumaigue in the bay of Antibes) and mixed before homogenization. Leaves were rinsed 3 times with distilled water, dried between two sheets of paper, frozen in liquid nitrogen and then stored at-80~ Preparation of mRNAs Most of classical methods so far described to isolate total RNA were ineffective with Posidonia tissues. In all cases, a large pellet which is suspected to contain large amounts of polysaccharides was obtained after adding ethanol to the extracted fraction. However, a modified hot borate method described by Wan and Wilkins (1994) allowed the preparation of non-degraded RNAs with a good yield and without contamination with polysaccharides. 1) Frozen posidonia tissues (15 g fresh weight) were pulverized to a free powder with a mortar and pestle in the presence of liquid nitrogen. The resulting powder was transferred into the hot (80~ extraction buffer (200 mM sodium borate deeahydrate [Borax], pH 9.0) containing: - EGTA - SDS - sodium deoxycholate - DTT - Polyvinylpyrrolidone [Mr 40,000] - Nonidet-40
30 mM 1.0% 1.0% 10 mM 2.0% 0.5%
Tissues (1 gr fresh weight) were extracted in 5 mL buffer.
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2) Tissues were ft~her homogenized for 2x 1 min using an Ultra-Turrax mixer. 3) Proteins were then digested by proteinase K (0.5 mg/mL extraction buffer) for 1.5 hours at 42~ with mild agitation on a rotary shaker. 4) RNAs were selectively precipitated in the presence of 2.0 M LiC1. 5) RNA pellets (from 1 gr tissues) were resuspended in 2 mL 10 mM Tris-HCl, pH 7.5. Salt-insoluble material was washed off in the presence of 200 mM potassium acetate pH 5.5 and precipitated again with 2.5 vol. Ethanol a t - 2 0 ~ The RNA pellet was washed with 70% ethanol and then resuspended in sterile distilled water.
Preparation of polyA+ RNAs Polyadenylated RNAs were purified on oligo dT cellulose column.
Construction of a eDNA library A eDNA library from posidonia tissues was constructed in the vector ~ZAPII (Stratagene), starting from 5 gg polyadenylated RNA according to the manufacturer' s instructions. The library was sized over 500 bp and the titter was 5.5 x 106 pfu/laL
Isolation of the cDNA encoding CA4H (CYP73) from posidonia The posidonia eDNA library was screened with a 32p CA4H eDNA from H. tuberosus corresponding to its coding sequence under low stringency conditions (hybridization at 55~ in 5 x SSC, washing at 45~ in 0.2 x SSC). An average of 35 positive clones were isolated among 50000 recombinants, suggesting that CA4H was about 0.1% of total proteins. Comparison of the nucleotidic sequence of the posidonia CA4H with sequences of other CA4H eDNA from several species shows more than 80% homology with the gene encoding CA4H from Poplar.
REFERENCES I. J.P. Sala'On and C. Helvig. Cytochrome P450-Dependent Oxidation of Fatty Acids. Drug Metabolism and Drug Interactions, 12 (1995) 261-283 2. J.F. Narbonne, P. Garrigues, D. Ribera, C. Raoux, A. Mathieu, P. Lemaire, J.P. Sala'0n and M. Lafaurie. Mixed-function oxygenase enzymes as tool for pollution monitoring: Field studies on the French coast of the Mediterranean Sea. Comp. Biochem. Physiol., 100c (1991), 37-42 3. D. Hamoutene, A. Mathieu, J.P. Sala'0n and M. Lafaurie. Mise en ~vidence de l'activit~ 6thoxyr6sorufine O-d~6thylase (EROD) chez la Posidonie : Posidonia oceanica (L.) vat. Delile. Oc6anis, VoL 18, Fasc. 2 (I 992) 199-206
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4. D. Hamoutene, A. Mathieu, P. Hofinann, J.P. Salaiin and M. Lafaurie. Preparation and characterization of subcellular fractions suitable for studies of xenobiotic metabolism from leave sheaths of a marine seagrass: Posidonia oceanica L. Delile. Mar. Environ. Res., 39 (1995) 249253. 5. C.Y. Wan and T.A. Wilkins. A modified hot borate method significantly enhances the yeld of high-quality RNA from cotton (Gossypitma hirsutmn, L.). Anal.Biochem. 223 (1994) 7-12
Technical Annex (Chapter 19) Development of Biomarkers from seagrass Posidonia oceanica A. Schoendorf, I. Benveniste, J.P. Salaiin Institut de Biologie Mol6culaire des Plantes, CNRS-UPR 406, 28 rue Goethe, 67083 Strasbourg Cedex, France
Material White (non chlorophyllous) and green (chlorophyllous) parts of Posidonia oceanica leaves (100g fresh weight) were collected from polluted area of the French Riviera (Nice, Monaco, La Foumaigue in the bay of Antibes) and mixed before homogenization. Leaves were rinsed 3 times with distilled water, dried between two sheets of paper, frozen in liquid nitrogen and then stored at-80~ Preparation of mRNAs Most of classical methods so far described to isolate total RNA were ineffective with Posidonia tissues. In all cases, a large pellet which is suspected to contain large amounts of polysaccharides was obtained after adding ethanol to the extracted fraction. However, a modified hot borate method described by Wan and Wilkins (1994) allowed the preparation of non-degraded RNAs with a good yield and without contamination with polysaccharides. 1) Frozen posidonia tissues (15 g fresh weight) were pulverized to a free powder with a mortar and pestle in the presence of liquid nitrogen. The resulting powder was transferred into the hot (80~ extraction buffer (200 mM sodium borate deeahydrate [Borax], pH 9.0) containing: - EGTA - SDS - sodium deoxycholate - DTT - Polyvinylpyrrolidone [Mr 40,000] - Nonidet-40
30 mM 1.0% 1.0% 10 mM 2.0% 0.5%
Tissues (1 gr fresh weight) were extracted in 5 mL buffer.
540
2) Tissues were ft~her homogenized for 2x 1 min using an Ultra-Turrax mixer. 3) Proteins were then digested by proteinase K (0.5 mg/mL extraction buffer) for 1.5 hours at 42~ with mild agitation on a rotary shaker. 4) RNAs were selectively precipitated in the presence of 2.0 M LiC1. 5) RNA pellets (from 1 gr tissues) were resuspended in 2 mL 10 mM Tris-HCl, pH 7.5. Salt-insoluble material was washed off in the presence of 200 mM potassium acetate pH 5.5 and precipitated again with 2.5 vol. Ethanol a t - 2 0 ~ The RNA pellet was washed with 70% ethanol and then resuspended in sterile distilled water.
Preparation of polyA+ RNAs Polyadenylated RNAs were purified on oligo dT cellulose column.
Construction of a eDNA library A eDNA library from posidonia tissues was constructed in the vector ~ZAPII (Stratagene), starting from 5 gg polyadenylated RNA according to the manufacturer' s instructions. The library was sized over 500 bp and the titter was 5.5 x 106 pfu/laL
Isolation of the cDNA encoding CA4H (CYP73) from posidonia The posidonia eDNA library was screened with a 32p CA4H eDNA from H. tuberosus corresponding to its coding sequence under low stringency conditions (hybridization at 55~ in 5 x SSC, washing at 45~ in 0.2 x SSC). An average of 35 positive clones were isolated among 50000 recombinants, suggesting that CA4H was about 0.1% of total proteins. Comparison of the nucleotidic sequence of the posidonia CA4H with sequences of other CA4H eDNA from several species shows more than 80% homology with the gene encoding CA4H from Poplar.
REFERENCES I. J.P. Sala'On and C. Helvig. Cytochrome P450-Dependent Oxidation of Fatty Acids. Drug Metabolism and Drug Interactions, 12 (1995) 261-283 2. J.F. Narbonne, P. Garrigues, D. Ribera, C. Raoux, A. Mathieu, P. Lemaire, J.P. Sala'0n and M. Lafaurie. Mixed-function oxygenase enzymes as tool for pollution monitoring: Field studies on the French coast of the Mediterranean Sea. Comp. Biochem. Physiol., 100c (1991), 37-42 3. D. Hamoutene, A. Mathieu, J.P. Sala'0n and M. Lafaurie. Mise en ~vidence de l'activit~ 6thoxyr6sorufine O-d~6thylase (EROD) chez la Posidonie : Posidonia oceanica (L.) vat. Delile. Oc6anis, VoL 18, Fasc. 2 (I 992) 199-206
541
4. D. Hamoutene, A. Mathieu, P. Hofinann, J.P. Salaiin and M. Lafaurie. Preparation and characterization of subcellular fractions suitable for studies of xenobiotic metabolism from leave sheaths of a marine seagrass: Posidonia oceanica L. Delile. Mar. Environ. Res., 39 (1995) 249253. 5. C.Y. Wan and T.A. Wilkins. A modified hot borate method significantly enhances the yeld of high-quality RNA from cotton (Gossypitma hirsutmn, L.). Anal.Biochem. 223 (1994) 7-12