Cold-induced expression of Δ9-desaturase in carp by transcriptional and posttranslational mechanisms

Science

Volumn 271, Issue 5250, 1996, Pages 815-818

  Tiku, P E ^a   Gracey, A Y ^a   Macartney, A I ^a   Beynon, R J ^b   Cossins, A R ^a  

^a UNIVERSITY OF LIVERPOOL   (United Kingdom)

^b UNIVERSITY OF MANCHESTER   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

ACYL COENZYME A DESATURASE; GLYCEROPHOSPHOLIPID;

ANIMAL TISSUE; ARTICLE; BODY TEMPERATURE; CARP; COLD; ENZYME ACTIVATION; ENZYME ACTIVITY; GENE EXPRESSION REGULATION; GENETIC TRANSCRIPTION; LIVER MICROSOME; MEMBRANE FLUIDITY; NONHUMAN; PRIORITY JOURNAL;

ANIMALIA; CYPRINUS CARPIO;

EID: 0029671331     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5250.815     Document Type: Article

References (17)

1. A R. Cossins, Ed., Temperature Adaptation of Biological Membranes (Portland Press, London, 1994).
2. J. R. Hazel, Annu. Rev Physiol. 57, 19 (1995)
3. M Schunke and E Wodtke, Biochim. Biophys. Acta 734, 70 (1983).
4. A. I. Macartney, B Maresca, A. R Cossins, in (1), pp. 129-139.
5. Carp were maintained in 2000-liter aquaria at 30°C for at least 3 months under a 12-hour day length and fed with a commercial trout diet (Mainstream expanded pellets; B.P Nutrition, Northwich, Cheshire, UK). They were gradually cooled over a 3-day period from 30°C to 23°C on day 1, 17°C on day 2, and 10°C on day 3 (3) and were maintained at 10°C for the remainder of the experiment Cooling was at 1°C per hour to the desired temperature and was then held constant for the remainder of the day At intervals, the hepatic postmitochondrial microsomal membrane fraction was isolated by differential centrifugation and recovered by centrifugation at 100,000g for 60 min. Lipids were isolated and phospholipid fractions were separated by thin-layer chromatography and transmethylated as described [A. R. Cossins and C L. Prosser, Biochim. Biophys Acta 687, 303 (1982)] The resulting methyl esters were analyzed by capillary gas chromatography (25 m × 0.25 mm; FFAP, Phase Separations, Queensferry, Clwyd, UK). Molecular species were analyzed by high-performance liquid chromatography (HPLC) separation of dinitrobenzene derivatives as described [H Takamura, H. Narita, R. Urade, M Kito, Lipids 21, 356 (1986)].
6. Carp were maintained in 2000-liter aquaria at 30°C for at least 3 months under a 12-hour day length and fed with a commercial trout diet (Mainstream expanded pellets; B.P Nutrition, Northwich, Cheshire, UK). They were gradually cooled over a 3-day period from 30°C to 23°C on day 1, 17°C on day 2, and 10°C on day 3 (3) and were maintained at 10°C for the remainder of the experiment Cooling was at 1°C per hour to the desired temperature and was then held constant for the remainder of the day At intervals, the hepatic postmitochondrial microsomal membrane fraction was isolated by differential centrifugation and recovered by centrifugation at 100,000g for 60 min. Lipids were isolated and phospholipid fractions were separated by thin-layer chromatography and transmethylated as described [A. R. Cossins and C L. Prosser, Biochim. Biophys Acta 687, 303 (1982)] The resulting methyl esters were analyzed by capillary gas chromatography (25 m × 0.25 mm; FFAP, Phase Separations, Queensferry, Clwyd, UK). Molecular species were analyzed by high-performance liquid chromatography (HPLC) separation of dinitrobenzene derivatives as described [H Takamura, H. Narita, R. Urade, M Kito, Lipids 21, 356 (1986)].
7. H. G. Enoch, A. Catala, P. Strittmatter, J Biol Chem. 251, 5095 (1976).
8. P Strittmatter, M A Thiede, C S. Hackett, J Ozols, J. Biol. Chem. 263, 2532 (1988).
9. +] RNA selected and separated by electrophoresis in 1% agarose-formaldehyde followed by capillary transfer to polyvinylidene difluonde (PVDF) membrane [J. Sambrook, E. F. Fntsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, vol 1)]. Blots were hybridized to the full-length cDNA insert from pcDsL7 at 42°C with the same medium as detailed above with highly stringent posthybridization washes according to manufacturer's (Millipore) instructions The hybridization product was 2.7 kb (P E Tiku, data not shown). Unidirectional deletion subclones of pcDsL7 were generated by exonudease III digestion (Erase-a-base, Promega) and autosequenced (Applied Biosystems model 373A) with universal pUC/M13 primers The full sequence was confirmed by autosequencing of the anticoding strand as having 2652 bp with an ORF of 879 bp, with a 520-bp 5′ untranslated region (UTR) and a 1253-bp 3′ UTR (GenBank, accession number CCU31864), Translation of this ORF indicated a polypeptide of 292 amino acid residues and a calculated molecular mass of 33 65 kD. For the clustal alignment (Fig. 2B), we used an identity residue weight table and DNAStar Lasergene software Amino acid sequences were obtained from cDNA sequences from GenBank and EMBL: rat (accession number J02585), tick (U03281), and yeast (J05676).
10. +] RNA selected and separated by electrophoresis in 1% agarose-formaldehyde followed by capillary transfer to polyvinylidene difluonde (PVDF) membrane [J. Sambrook, E. F. Fntsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, vol 1)]. Blots were hybridized to the full-length cDNA insert from pcDsL7 at 42°C with the same medium as detailed above with highly stringent posthybridization washes according to manufacturer's (Millipore) instructions The hybridization product was 2.7 kb (P E Tiku, data not shown). Unidirectional deletion subclones of pcDsL7 were generated by exonudease III digestion (Erase-a-base, Promega) and autosequenced (Applied Biosystems model 373A) with universal pUC/M13 primers The full sequence was confirmed by autosequencing of the anticoding strand as having 2652 bp with an ORF of 879 bp, with a 520-bp 5′ untranslated region (UTR) and a 1253-bp 3′ UTR (GenBank, accession number CCU31864), Translation of this ORF indicated a polypeptide of 292 amino acid residues and a calculated molecular mass of 33 65 kD. For the clustal alignment (Fig. 2B), we used an identity residue weight table and DNAStar Lasergene software Amino acid sequences were obtained from cDNA sequences from GenBank and EMBL: rat (accession number J02585), tick (U03281), and yeast (J05676).
11. 32P uridine 5′-triphosphate. The β-actin template was derived by subcloning the Sac I-Xba 1 fragment from CA16-Sal I [Z. Liu et al., DNA Sequence-J DNA Sequencing and Mapping 1. 125 (1990)] into the Promega vector pGEM7Zf(+). The recombinant clone pG7ccactb was digested with Hpa I to generate a template from which a probe of -700 nucleotides (nt) was synthesized. Unidirectionally deleted pcDsL7 resulted in subclone pcDsL7D*, which had all of the 5′ UTR deleted. This was digested with Xho I and protected with α-phosphorothioate deoxynucleotide triphosphates. Unidirectional deletion of Hind III-digested and protected pcDsL7D* resulted in subclone pcDsL7d4, which was digested with Nae I to give an 830-bp fragment, which was then used as a template for producing the 300-nt desaturase antisense probe The 109-nt anti-18S rRNA probe was synthesized from pT7 18S RNA (Ambion) Nuclei were isolated from carp liver, RNA synthesized with unlabeled nucleotide triphosphates [W. F. Marzluff and R C, C Huang, in Transcription and Translation: A Practical Approach, B. D. Hames and S. J. Higgins, Eds (IRL Press, Oxford, 1984), pp. 89-130] and 10 μg of RNA analyzed by RPA with the antisense desaturase probe.
12. 32P uridine 5′-triphosphate. The β-actin template was derived by subcloning the Sac I-Xba 1 fragment from CA16-Sal I [Z. Liu et al., DNA Sequence-J DNA Sequencing and Mapping 1. 125 (1990)] into the Promega vector pGEM7Zf(+). The recombinant clone pG7ccactb was digested with Hpa I to generate a template from which a probe of -700 nucleotides (nt) was synthesized. Unidirectionally deleted pcDsL7 resulted in subclone pcDsL7D*, which had all of the 5′ UTR deleted. This was digested with Xho I and protected with α-phosphorothioate deoxynucleotide triphosphates. Unidirectional deletion of Hind III-digested and protected pcDsL7D* resulted in subclone pcDsL7d4, which was digested with Nae I to give an 830-bp fragment, which was then used as a template for producing the 300-nt desaturase antisense probe The 109-nt anti-18S rRNA probe was synthesized from pT7 18S RNA (Ambion) Nuclei were isolated from carp liver, RNA synthesized with unlabeled nucleotide triphosphates [W. F. Marzluff and R C, C Huang, in Transcription and Translation: A Practical Approach, B. D. Hames and S. J. Higgins, Eds (IRL Press, Oxford, 1984), pp. 89-130] and 10 μg of RNA analyzed by RPA with the antisense desaturase probe.
13. A I Macartney, P Tiku, A. R. Cossins, in preparation.
14. E. Wodtke and A. R. Cossins, Biochim. Biophys. Acta 1064, 343 (1991)
15. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E. Glu, F, Phe; G, Gly; H, His; I, Ile, K, Lys; L, Leu, M, Met; N, Asn; P, Pro; Q, Gln; R, Arg, S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr
16. 14C-labeled substrate [J. B. Leifkowith, Biochim. Biophys. Acta 1044, 13 (1990)].
17. We thank E Fodor for HPLC analyses of molecular species; P. Strittmatter for providing the antibody to rat desaturase and the cloned rat cDNA, pDs3-358, P B Hackett for the carp β-actin clone CA16-Sal I, and M Caddick, B. Maresca, and J. Crampton for helpful discussions. Supported by a grant and postgraduate support from the Natural Environmental Research Council (UK).