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Stearoyl-CoA desaturase mRNA is transcribed from a single gene in the ovine genome1
Biochimica et Biophysica Acta 1391 Ž1998. 145–156
Stearoyl-CoA desaturase mRNA is transcribed from a single gene in the ovine genome 1 Richard J. Ward a,b, Maureen T. Travers a , Sion E. Richards b, Richard G. Vernon a , Andrew M. Salter b, Peter J. Buttery b, Michael C. Barber a,) a
b
Hannah Research Institute, Ayr, KA6 5HL, UK Department of Applied Biochemistry and Food Science, Nutritional Biochemistry Laboratory, UniÕersity of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK Received 23 September 1997; revised 25 November 1997; accepted 11 December 1997
Abstract Clones corresponding to ovine stearoyl-CoA desaturase ŽSCD. cDNA were isolated from an adipose tissue cDNA library. All of these clones represented a single mRNA species as judged by restriction fragment and DNA sequence analysis. RNase protection analysis demonstrated that this SCD transcript is highly expressed in adipose tissue and liver, and in the mammary gland of lactating animals. A lower level of expression was detectable in a variety of other tissues including brain. Levels of the SCD transcript were decreased in adipose tissue during lactation, and this appears to be related to a marked decline in serum insulin and insulin-responsiveness of the tissue. Southern analysis of ovine and mouse genomic DNA demonstrated that the ovine SCD cDNA hybridised in a manner consistent with a single gene for SCD in ovine DNA; mouse genomic DNA produced a pattern of hybridisation consistent with the previously characterised mouse SCD-1 and SCD-2 genes. Three ovine cosmids were isolated that comprised the restriction fragments predicted by the genomic Southern analysis. The ovine SCD gene was predicted to be encompassed within a 23 kbp region that was present in all three cosmids. These results demonstrate that SCD is transcribed from a single gene in the ovine genome and this gene is insulin-responsive in ovine adipose tissue. q 1998 Elsevier Science B.V. Keywords: Stearoyl-CoA desaturase; cDNA; Gene; Adipose tissue; Lactation
1. Introduction Abbreviations: ACC, acetyl-CoA carboxylase; cDNA, complementary deoxyribonucleic acid; kbŽp., kilobase Žpairs.; MMLV, murine moloney leukemia virus; ORF, open reading frame; RNase, ribonuclease; SCD, stearoyl-CoA desaturase; UTR, untranslated region ) C orresponding author. Fax: q 44-1292-674003; E-mail: [email protected] 1 The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AJ001048.
Stearoyl-CoA desaturase ŽSCD. catalyses the oxidation of palmitoyl-CoA and stearoyl-CoA at the D 9 position to form the monounsaturated fatty acyl-CoA esters, palmitoeyl-CoA and oleoyl-CoA, respectively; stearoyl-CoA is the preferred substrate w1x. In rodents, SCD enzyme activity is the result of transcription from two related genes Ž SCD-1 and SCD-2. in a
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 2 1 0 - 5
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tissue-specific fashion, each of which encode a highly similar functional enzyme w2,3x. SCD-1 is mainly expressed in adipose tissue and liver of rodents; SCD-2 is principally expressed in the brain w2,3x. The importance of SCD is indicated by the fact that the proportion of saturated and monounsaturated fatty acids of a cell are reflected, not only in the composition of the triacylglycerol, but also in the membrane phospholipid. The ratio of stearic acid to oleic acid incorporated into phospholipids has a significant influence on membrane fluidity, and alteration in this ratio has been implicated in a variety of disease states such as cancer, diabetes, obesity, vascular and coronary heart disease w4–9x. With respect to vascular and coronary disease, a major risk factor for progression is the consumption of high levels of saturated fat in the diet w10x, principally from the meat of domestic ruminants. Ruminant animals in general, and sheep in particular have relatively high levels of saturated to monounsaturated fatty acids in their lipids w11x. This arises from a variety of factors. Dietary fatty acids Žwhich are usually mostly polyunsaturated. are hydrogenated by rumen microorganisms; hence, a large proportion of the dietary fatty acids absorbed are saturated w11x. Also, as ruminant diets have a relatively low fat content, there is a high rate of fatty acid synthesis in adipose tissue, the principal products of which are palmitoyl-CoA and stearoyl-CoA. Ruminant tissues have SCD activity but little is known about its regulation. As increasing the degree of desaturation of the lipid of ruminant tissues, especially adipose tissue and milk triacylglycerol, is likely to be beneficial from the point of view of human nutrition, we have investigated the SCD system of ovine tissues.
2. Materials and methods 2.1. Animals and tissue culture Sheep were Finn–Dorset crossbred animals, fed on hay and cereals for at least four weeks before slaughter w12x. Animals were used either as control Ž nonpregnant non-lactating., pregnant or lactating ewes or wethers Žsix month-old castrated-males.. Female sheep were all 3–5 years old and multiparous. Pregnant animals were used between 100–105 days of
gestation and lactating ewes, suckling at least two lambs, were used at about day 18 of lactation. Animals were anaesthetised, exsanguinated and samples of subcutaneous adipose tissue obtained, snap frozen and stored in liquid nitrogen. Subcutaneous adipose tissue removed aseptically from lactating animals was used to prepare explants. These were maintained in culture in Medium 199 w12x for periods of up to 72 h in the presence or absence of insulin Ž 17 nM. plus dexamethasone Ž 10 nM.. Fresh explants of adipose tissue and explants after culture were snap-frozen and stored in liquid nitrogen. 2.2. Isolation of cDNA clones An ovine adipose cDNA library was constructed in lgt-11 using cDNA prepared from reverse-transcription of adipose tissue RNA w13x by MMLV reverse transcriptase using oligo-dT as a primer. The cDNA was double-stranded and then ligated to EcoRI–NotI adaptors Ž Pharmacia Biotech. prior to ligation into EcoRI restricted and dephosphorylated lgt-11 vector by standard methods w14x. The library was plated on Y1089 host bacteria and replica-plated onto nitrocellulose filters. The filters were hybridised with a rat SCD-1 cDNA corresponding to the coding region of the mRNA w15x radioactively labelled to high-specific activity by the random-priming method w16x. After several rounds of screening, positive recombinant plaques were purified to homogeneity, and the resulting bacteriophage DNA was isolated by standard methods w17x. 2.3. Isolation of genomic clones High molecular weight genomic DNA was isolated from sheep spleen by standard methods w18x. The DNA was partially digested with Sau3AI restriction endonuclease and a fraction corresponding to a size of 33 to 50 kbp was isolated by sucrose-density gradient centrifugation w19x. The resulting DNA was then ligated into the SuperCos I cosmid vector ŽStratagene. and packaged using Gigapack II gold extracts ŽStratagene.. The library corresponding to 8 = 10 5 individual recombinants was plated on the XL1-blue MR host. Aliquots of the library were replica-plated and screened with a radioactively labelled w16x ovine SCD cDNA Ž clone 3A, see below. .
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Cosmids were purified to homogeneity by several rounds of screening. Cosmid DNA was isolated by standard plasmid protocols. Southern analysis of genomic DNA and cosmids was performed using standard methods w14x; filters were washed at 0.2 = SSCr0.1% Žwrv. sodium dodecyl sulfate at 558C Ž20 = SSC s 3 M NaCl, 0.3 M sodium citrate. . 2.4. DNA sequencing and computer analysis Inserts from lgt-11 recombinant bacteriophage were excised with NotI and subcloned in pGEM ŽPromega. plasmid vectors. DNA sequencing was performed by the dideoxy-chain termination method w20x using w a- 35 SxdATP and Taq DNA polymerase with M13rpUC forward and reverse sequencing primers or gene-specific primers ŽCustom-synthesised by Cruachem, Glasgow. . DNA sequence was analysed for similarity using the GCG package Ž University of Madison, WI.. 2.5. RNase protection assay An RNase protection assay was developed using a fragment of the ovine SCD cDNA as a probe. A 392 nucleotide NcoI–EcoRV fragment of clone 1D was subcloned into pGEM-7zfq and used to generate an antisense transcript using SP6 RNA polymerase and a- 32 P CTP w21x. Tissue samples were powdered in a mortar and pestle using liquid nitrogen and homogenised in 5 M guanidinium isothiocyanate, 100 mM EDTA, pH 7.0, using a constant tissue to volume ratio. Aliquots Ž40 m l. of these were hybridised to the antisense transcript and subsequently digested using RNaseArRNase T1 and proteinase K w22x. In addition, various amounts of sense transcript were hybridised to the antisense transcript as molar standards to allow SCD transcript abundance to be determined. After extraction with phenol and chloroform, the samples were precipitated twice and rinsed with 80% Žvrv. alcohol. Samples were dried, resuspended, and after denaturing in formamide loading buffer at 858C, resolved on a 6% Ž wrv. acrylamider8 M urea sequencing gel w23x. After drying, the gels were exposed to a Kodak phosphor screen overnight. The resulting images were scanned using a Molecular Dynamics phosphorimager 445 SI and the volume of individual bands obtained using ImageQuant software. Homogenate DNA content was assayed by the
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method of Labarca and Paigen w24x after extraction of the same tissue homogenate with four volumes of water-saturated chloroform. SCD mRNA was expressed as transcripts per cell by dividing the number of transcripts per volume of homogenate by the cell number per volume of homogenate Žobtained by dividing the DNA content in pg by the diploid DNA content of a cell, assuming a cell contains 7 pg of genomic DNA w25x.. 2.6. Preparation of adipocytes and stromoÕascular cells Adipocytes were prepared by collagenase digestion of omental adipose tissue, essentially as described previously w26x. Adipocytes were recovered by flotation, after which the stromovascular cells in the infranatant were pelleted by centrifugation at 4200 = g for 10 min. The adipocytes and stromovascular cells were resuspended in 5 M guanidine isothiocyanate, 100 mM EDTA pH 7.0, and portions of the cell homogenates were used in the SCD mRNA RNase protection assay and for DNA determination as described above. 2.7. Statistical analysis Data was analysed by one-way ANOVA. 3. Results 3.1. Isolation of cDNA clones from the oÕine omental adipose tissue library For the initial screening of the cDNA library 2 = 10 5 recombinant plaques were screened with a 1.2kbp cDNA insert corresponding to rat SCD-1 w15x. The primary screen resulted in 12 positives, of which nine were purified to homogeneity after successive rounds of screening. The cDNA inserts from the l recombinants were subsequently sub-cloned into plasmid pGEM-7zfŽq. and initially characterised by restriction endonuclease mapping and DNA sequencing ŽFig. 1.. Restriction endonuclease mapping of the recombinants essentially described one species of cDNA that was subsequently confirmed by complete or partial DNA sequencing of the nine recombinants. The sequence data from all the recombinants indi-
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Fig. 1. Strategy for cloning and sequencing ovine SCD cDNAs. An oligo-dT sheep adipose cDNA library was screened with a rat SCD-1 cDNA insert w15x. The resulting ovine clones are shown with reference to the structure of the rat SCD-1 mRNA. The shaded box denotes the open-reading frame of rat SCD-1. Restriction enzyme abbreviations ŽA s ApaI; E s EcoRV; H s HindIII; Nc s NcoI; Ns s NsiI; P s PstI; S s SacI; Xa s Xba; Xo s XhoI.. The black arrowheads and dotted lines show the direction and extent of individual sequencing reactions. The black box denotes the 392 nucleotide Nc–E fragment used in the RNase protection assay ŽFigs. 4 and 8..
cated only one open reading frame Ž ORF. for the SCD mRNA species, principally expressed in ovine adipose tissue. The complete coding sequence contains 1077 nucleotides encoding a protein of 359 amino acids with a calculated Mr of 41 742 ŽFig. 2.. The largest recombinant Ž3D. to contain a complete ORF approximated to 3.4 kbp which is significantly smaller than the estimated size of 5 kb from Northern blotting of ovine adipose RNA Žresults not shown.. Furthermore, this recombinant did not possess a polyA tract nor a polyadenylation motif at its 3X terminius despite being isolated from an oligo-dT primed cDNA library. This suggests that this clone and the other SCD recombinants isolated from the cDNA library resulted from non-poly-A tail priming of mRNA. In rodents, SCD activity is the result of transcription from two related genes ŽSCD-1 and -2. in a tissue-specific fashion. Complementary DNAs corre-
sponding to the complete ORF for rat w15x and mouse SCD-1 w2x and mouse SCD-2 w3x have been previously described; partial sequences have also been derived for rat SCD-2 w28x and human SCD w29x. In addition cDNAs related to these SCDs have been isolated for the sheep tick Amblyomma w30x, the protozoan Tetrahymena w31x and yeast w32x. Comparison of these amino acid sequences for the higher eukaryotes Žusing the GCG Homologies algorithm. demonstrate that the ovine SCD amino acid sequence has 89–93% identity with mouse, rat and human SCDs, with a somewhat lower identity with Amblyomma SCD Ž74%. and the other lower eukaryotes Žresults not shown.. However, it is not possible to ascertain whether the ovine SCD has greater identity with mouse SCD-1 or with mouse SCD-2, as both appear to diverge approximately equally from the ovine SCD Ž89% identity. .
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Fig. 2. The nucleotide sequence of ovine SCD cDNA. The codon for the initiator methionine and the stop codon are underlined. The open-reading frame is denoted by three letter abbreviations for the amino acids. The His residues indicated by bold script correspond to eight histidines spatially conserved in the family of membrane desaturases which are thought to act as iron-centres in the active site of SCD w27x.
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Sequence comparisons of the 5X UTRs of the SCDs demonstrates that the ovine SCD 5X UTR exhibits a higher identity with rat and mouse SCD-1 Žboth 64%. as opposed to the corresponding SCD-2s Žboth 58%.. The similarity between the ovine SCD and the rodent SCDs is particularly marked around the translation initiation codon and then subsequently diverges towards the 5X termini of the respective SCD cDNAs ŽFig. 3. . Conversely, comparison of 695 nucleotides downstream of the termination codon Ž clone 1D, Fig. 1. in the ovine SCD mRNA 3X UTR with the corresponding rodent SCD cDNAs demonstrates a
55% identity with mouse SCD-2, which is significantly higher than the identity with rat and mouse SCD-1s Žboth 47%.. Such comparison is interesting, as similar analysis reveals a low identity in the 3X UTR between mouse SCD-1 and mouse SCD-2 Ž 44%. and a relatively high identity between mouse and rat SCD-1 Ž81%.. 3.2. Expression of SCD gene in oÕine tissues To determine the relative expression of the SCD transcript in a variety of ovine tissues, an RNase
Fig. 3. Multiple alignment of the 5X untranslated region of ovine Žov. SCD cDNA with those of mouse Žm. and rat Žr. SCD-1 and SCD-2 cDNAs. The alignment was performed with the CLUSTALV program. Black box areas denote sequence conservation. Numbers at the right hand margin indicate the nucleotide residues.
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protection assay using a 392 nucleotide NcoI–EcoRV fragment of clone 1D ŽFig. 1. was established. An RNase protection assay also has the advantage of being able to evaluate the number of copies of the SCD transcript in the ‘average’ cell comprising the tissue of interest, assuming a cell contains approximately 7 pg of genomic DNA w25x. The results of such analysis is shown in Fig. 4A,B. This demonstrates that although the SCD transcript was detected in all tissues examined from wether sheep, the highest levels of expression per volume of tissue homogenate appeared to be in liver and in the mammary gland of lactating animals Ž Fig. 4A. . However, when the DNA content of the homogenate was taken into account and the results expressed as SCD transcripts per cell, it can be seen that the highest level of expression, approximating to 100–250 transcripts per cell, was observed in adipose tissue and liver ŽFig. 4B., tissues that are major sites of triacylglycerol synthesis. The apparent discrepancy between the relatively low level of SCD expression in the adipose tissue homogenate Ž Fig. 4A. , and the markedly higher expression when the data is expressed as transcriptsrcell ŽFig. 4B. , is due to adipose tissue homogenates having a low DNA concentration, due in part to approximately 70% of their tissue volume comprising lipid. Fractionation of adipose tissue into the stromovascular cells Žendothelial cells and fibroblasts. and an adipocyte-enriched fraction demonstrated that expression of SCD mRNA in the adipocyte fraction was 140-fold higher per cell than in the stromovascular fraction Žresults not shown.. This implies that adipocytes, which comprise only 5–10% of the cells per unit tissue w33x, are the principal site of SCD gene expression in adipose tissue. Of the remaining tissues, the range of expression varied from 1 transcript per cell in pancreas to 31.5 transcripts per cell in muscle; SCD mRNA was readily detected in ovine brain with approximately 10 transcripts per cell. Additionally, high levels of SCD mRNA were also found in the mammary gland from lactating sheep ŽFig. 4B. . 3.3. OÕine SCD is transcribed from a single gene in the oÕine genome Southern analysis of ovine genomic DNA restricted with a variety of restriction endonucleases
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Fig. 4. Determination of SCD mRNA in ovine tissues by RNase protection assay. ŽA. Homogenates were prepared from various ovine tissues and these were used in a RNase protection assay with antisense SCD probe resulting in a 392 nucleotide protected fragment. The band of 467 nucleotides is undigested antisense SCD probe and represents approximately 0.1% of the probe added to each assay. ŽB. The 392 nucleotide protected fragments were quantitated using a phosphorimager with ImageQuant software. DNA content of the homogenates was determined and SCD mRNA was expressed as the number of transcriptsrcell by dividing the number of transcripts per volume of homogenate by the cell number per volume homogenate Žobtained by dividing the homogenate DNA content in pg by the diploid DNA content of a cell, assuming a cell contains 7 pg of genomic DNA w25x.: Br s Brain; K s Kidney; H s Heart; M s Muscle; P s Pancreas; Lu s Lung; L s Liver; SpsSpleen; Ad s Adipose tissue; LMG s lactating mammary gland. Values are means"S.E.M. from three animals.
and then hybridised with a SCD cDNA corresponding to the ORF Žclone 3A. generated a relatively simple hybridisation pattern Ž Fig. 5A. ; a variety of
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Fig. 5. Southern analysis of genomic DNA isolated from ŽA. sheep and ŽB. mouse and then hybridised with ovine SCD cDNA. Twenty m g of genomic DNA was digested with restriction enzymes as indicated and then separated on a 0.8% Žwrv. agarose gel, which was blotted onto a nylon membrane. This was hybridised with ovine SCD cDNA insert Žclone 3A.. Numbers at the left hand margin in ŽA. indicate the position of DNA markers. Restriction fragments in the mouse genomic DNA ŽB. that hybridised with ovine SCD cDNA are indicated with reference to fragments that have been reported to hybridise with the homologous murine SCD-1 cDNA w2x and that have been identified in the murine SCD-1 w2x and SCD-2 w3x genes.
enzymes Ž EcoRI, BamHI, SacI. principally generated a single large hybridisation fragment Ž approximately 21 kbp in the case of EcoRI. whereas others Ž HindIII, Pst I. generated smaller multiple fragments. A HindIII site is present in the SCD ORF Ž corresponding to murine SCD exon 2. , and thus accounts for the two fragments generated by HindIII of 5 kbp and 8 kbp in Fig. 5A. By comparison, Southern analysis of mouse genomic DNA digested with EcoRI ŽFig. 5B. and then hybridised with the ovine SCD cDNA generates a more complex pattern of hybridisation; fragments of 14.0, 9.0, 4.5 and 2.2 kbp were generated. Fragments of similar size can be observed
in the murine SCD-1 and SCD-2 genes w2,3x. A similarly complex pattern of hybridisation was observed with mouse DNA digested with HindIII; fragments of similar size can be observed in the murine SCD-1 and SCD-2 genes w2,3x, although a complete restriction map for HindIII is not available for the mouse SCD-2 gene w3x. Screening of an ovine cosmid library Ž 4 = 10 5 colonies. with the SCD cDNA corresponding to the ORF Žclone 3A. resulted in the isolation of three clones Žcosmids 3, 7 and 8. . These cosmids were restriction-mapped and were demonstrated to correspond to an overlapping series of genomic DNA
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clones ŽFig. 6.. Ovine SCD cDNA clone 3A hybridised to a 21-kbp EcoRI fragment in all the cosmids. Additionally, each cosmid hybridised to SCD cDNAs corresponding to both the 5X UTR and the terminal portion of the 3X UTR, suggesting that the entire SCD transcriptional unit was represented within a genomic region that approximated to 23 kbp. This comprised the 21 kbp EcoRI fragment and a 2-kbp EcoRI fragment that hybridised with the 3X UTR cDNA ŽFig. 6.. The ends of each cosmid were distinct and comprised multiple EcoRI fragment repeats of 0.8 kbp. Furthermore, double-stranded DNA sequencing of each of the three cosmids using an oligonucleotide predicted to occur within exon 2 Žby analogy with the structures of the two murine genes. as a primer generated the same nucleotide sequence; the 3X portion of exon 2 was conserved as predicted and the sequence of the 5X region of the adjacent intron was identical in the three cosmid clones Ž results not shown.. Digestion of the cosmids with a variety of restriction enzymes used in the Southern analysis of ovine genomic DNA Ž Fig. 5A. generated essentially the same pattern of hybridisation ŽFig. 7. . For example, digestion of each of the cosmids with HindIII generated the 5.0 and 8.0 kbp fragments that hybridised with clone 3A; digestion with Pst I produced fragments of 4.0, 2.5 and 0.7 kbp that hybridised with clone 3A and that were also present in the genomic DNA Ž Fig. 5A. . These results demonstrated that a single genomic unit corresponding to the ovine SCD gene had been isolated from the
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Fig. 7. Southern analysis of cosmid 3 demonstrating that restriction fragments in ovine genomic DNA that hybridised to SCD cDNA are present in the cosmid clone. One m g of cosmid DNA was digested with the complement of restriction enzymes used in Fig. 5A and Southern-blotted. This was hybridised with ovine SCD cDNA Žclone 3A.. Numbers at the left hand margin indicate the position of DNA markers.
cosmid library with a structure predicted by the Southern blotting of the restricted genomic DNA. 3.4. The SCD gene is repressed in adipose tissue during pregnancy and lactation Lactation results in a major decrease in the expression of the SCD gene in sub-cutaneous adipose tissue, being reduced from 678 " 271 mRNA copies per cell in non-pregnant non-lactating animals to 76 " 35 mRNA copies per cell during lactation Ž P - 0.05. ŽFig. 8. . A similar level of SCD gene repression was
Fig. 6. Restriction fragments corresponding to the ovine SCD gene are encompassed within three clones of the ovine cosmid library. Cosmids 3, 7 and 8 were digested with various restriction enzymes ŽEI s EcoRI; H s HindIII; S s SacI. and Southern blotted. The blot was sequentially hybridised with cDNA inserts corresponding to the coding region Žclone 3A., the 5X UTRrN-terminal coding region Žan ApaI–ApaI subclone of 3D. and the 3X UTR Ža NsiI–NcoI subclone of 3D.. The position of the fragments that hybridised with each of the cDNAs are indicated by the shaded boxes. Each of the cosmids comprised a 21-kb and a 2-kb EcoRI fragment that hybridised with one or more of the ovine SCD cDNAs. The ends of each cosmid were distinct and comprised EcoRI fragment repetitive elements that are not shown.
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4. Discussion
Fig. 8. Expression of SCD mRNA in adipose tissue during pregnancy and lactation and in explants from lactating sheep cultured with insulin plus dexamethasone. Homogenates were prepared from subcutaneous adipose tissue from non-pregnant non-lactating ŽC., 100–105 day pregnant ŽP. and lactating animals ŽL., and from sub-cutaneous adipose explants from lactating sheep at time zero Ž0. and after culture for 72 h with no hormones ŽNA. or with insulin plus dexamethasone ŽIqD.. These were used in an RNase protection assay with antisense SCD transcript resulting in a 392 nucleotide protected fragment. The protected fragments were quantitated using a phosphorimager with ImageQuant software. DNA content of the homogenates was determined and SCD mRNA was expressed as the number of transcriptsrcell, assuming a cell contains 7 pg of genomic DNA w25x. Values were analysed by ANOVA and are means"S.E.M. of seven animals; ) ) ) P - 0.001 compared with explants at zero time.
also evident in the sub-cutaneous adipose tissue depot of animals that were 100 days pregnant ŽFig. 8.. Preparation and culture of adipose tissue explants from the sub-cutaneous depot of lactating sheep in Medium 199 containing insulin plus dexamethasone for 72 h results in an approximate 15-fold increase in the level of SCD transcripts from 151 " 37 mRNA copies per cell in the freshly prepared explants to 2198 " 340 mRNA copies per cell at the end of culture with the added hormones Ž P - 0.001., approximately 3-fold higher than the level of SCD expression in the sub-cutaneous depot of non-pregnant non-lactating sheep ŽFig. 8. . Culture of adipose explants for 72 h in Medium 199 without added hormones resulted in a level of SCD gene expression similar to the freshly prepared explants.
In mice, SCD mRNA is transcribed from two evolutionary related genes, SCD-1 and SCD-2 w2,3x in a tissue-specific fashion. For example, SCD-1 is the major transcript in adipose tissue and liver w3x. SCD-1 does not appear to be expressed in brain; SCD transcripts in brain are derived from the SCD-2 gene. Conversely, the SCD-2 gene is not transcribed in liver and is only transcribed at low levels in adipose tissue w3x. In this present study, we have isolated nine independent clones corresponding to ovine SCD from an adipose tissue cDNA library; these clones correspond to the same molecular species as judged by restriction mapping and DNA sequence. In contrast to mouse SCD mRNA, this ovine adipose tissue SCD transcript appears to be widely expressed, though expression is highest in adipose tissue and liver and in the mammary gland of lactating animals. This SCD transcript is readily detectable in ovine brain. These differences in the expression profile of SCD transcripts in mice and sheep are unlikely to be attributed to assay sensitivity, as both ourselves and Kaestner et al. w3x used RNase protection assays to evaluate SCD gene expression. Southern analysis of ovine genomic DNA using a cDNA encompassing the SCD ORF demonstrated a relatively simple pattern of fragment hybridisation; a number of restriction enzymes generated a single hybridisation fragment. This contrasts with mouse genomic DNA in which mouse SCD-1 cDNA recognises multiple bands in the mouse genome even at high stringency, some of which can be accounted for by the mouse SCD-2 gene. Indeed, the SCD-2 gene was isolated from mouse genomic DNA libraries using the mouse SCD-1 cDNA w3x. Interestingly, ovine SCD cDNA hybridises with multiple fragments generated by a number of restriction endonucleases in the mouse genome; on the basis of size, these can be assigned to either the SCD-1 or the SCD-2 genes w2,3x. Thus, the relatively simple hybridisation pattern observed with ovine genomic DNA is consistent with there being a single copy of the SCD gene in the haploid ovine genome. Indeed, screening of an ovine cosmid library with SCD cDNA generated an overlapping series of clones corresponding to a single genomic region; this was confirmed by DNA sequencing of the region predicted to represent the
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boundary of the 2nd exon and the 2nd intron, and the presence of restriction fragments occurring within the cosmid clones that were predicted to occur in the SCD locus from the genomic Southern blots. Thus, it appears that the gene duplication that resulted in the SCD-1 and SCD-2 genes in mouse w2,3x and rat w15,28x is a relatively recent event in evolution, occurring after the species divergence of rodents and ruminants. It is not clear why rodents have evolved two SCD genes and sheep only one, although it could relate to the differing complexity of the tissue-specific control of lipid metabolism in each species. Rodents, in the wild at least, exhibit marked diurnal variations in food intake and have diets of variable fat content with the result that different tissues would have different temporal requirements for monounsaturated fatty acid; for example, brain would still have a need for monounsaturated fatty acids for myelin formation at times when fatty acid synthesis in adipose tissue is low w34x. Sheep tissues, on the other hand, tend to have a more constant supply of nutrients that are usually low in fatty acids, due to the nature of the diet and the buffering action of the rumen w35x. Lactation results in a profound change in the lipid synthetic activity of adipose tissue in sheep with a major decrease in the rate of fatty acid synthesis, and the activity of the key lipogenic enzyme acetyl-CoA carboxylase ŽACC.; there are also smaller decreases in the rate of fatty acid esterification and the activity of glycerol-3-phosphate acyltransferase and lipoprotein lipase w12,33,36,37x. The decrease in ACC activity is at least partly due to a fall in the amount of ACC mRNA w33x. The present study shows that there is also a fall in SCD mRNA with lactation. The changes in lipid synthetic activity in sheep adipose tissue as described above occurs around day 105 of pregnancy w33,36x at a time when demands for nutrients of the growing foetusŽes. and mammary glands are rapidly increasing. The amount of SCD mRNA is also decreased by day 105 of pregnancy. The major factors responsible for the decreased capacity for lipid synthesis in adipose tissue during late-pregnancy and during lactation are thought to include insulin and growth hormone, the serum concentrations of which decrease and increase respectively w36x, and insulin resistance of adipose tissue w37x. Maintenance of explants of adipose tissue from lactating sheep in culture for 72 h with insulin plus
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dexamethasone restores the rate of lipogenesis to levels found in non-lactating ewes, and increases the amount of ACC mRNA to levels in excess of those found in non-lactating ewes w33x; culture with insulin plus dexamethasone had a similar effect on the amount of SCD mRNA. This enhanced level of expression obtained during culture with insulin plus dexamethasone may be explained by a change in the balance of activators and repressors of the SCD and ACC genes resulting in a greater relief of repression in the in vitro system. This is likely to be achieved by the absence of negatively acting hormones, such as growth hormone that has been implicated in repressing ACC and fatty acid synthase gene expression in adipose tissue of pigs and rats, respectively w38–40x. The mechanism for the repression of the SCD gene, and how this is coordinated with suppression of lipogenesis is unknown, though as both parameters are relieved by insulin plus dexamethasone in vitro, suggests that attenuation of a common or related signalling event may be involved. Future consideration of the mechanism for the repression of the SCD gene in ovine adipose tissue during lactation and its subsequent de-repression in vitro will require characterisation of the promoter to elucidate the factors that regulate transcription of the gene. Recently, 4.3 kb of the mouse SCD-1 5X flanking sequence has been shown to be insulin-responsive in transient transfection of a mouse liver cell line, suggesting the presence of an insulin-response element within the transfected DNA w41x. DNA sequence comparison of the 5X flanking sequence of the ovine SCD gene with that of the mouse SCD-1 gene may aid in the identification of regulatory domains within each promoter.
5. Conclusion The activity of SCD in the adipose tissue of sheep and other ruminant animals is likely to be critical in determining the saturated fat content of meat. Any attempt at altering the fatty acid composition in favour of less saturated fatty acids may be dependent on manipulation of the expression of the SCD gene. Our findings of only a single gene in the ovine genome and that expression of the gene is under hormonal control may help in designing such strategies.
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Acknowledgements The authors thank Mrs. Mandy Vallance and Mrs. Helen Pollock for expert technical assistance. This work was funded by a Link grant from the Biotechnology and Biological Sciences Research Council and the Scottish Office Agriculture Environment and Fisheries Department. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AJ001048.
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Stearoyl-CoA desaturase mRNA is transcribed from a single gene in the ovine genome 1 Richard J. Ward a,b, Maureen T. Travers a , Sion E. Richards b, Richard G. Vernon a , Andrew M. Salter b, Peter J. Buttery b, Michael C. Barber a,) a
b
Hannah Research Institute, Ayr, KA6 5HL, UK Department of Applied Biochemistry and Food Science, Nutritional Biochemistry Laboratory, UniÕersity of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK Received 23 September 1997; revised 25 November 1997; accepted 11 December 1997
Abstract Clones corresponding to ovine stearoyl-CoA desaturase ŽSCD. cDNA were isolated from an adipose tissue cDNA library. All of these clones represented a single mRNA species as judged by restriction fragment and DNA sequence analysis. RNase protection analysis demonstrated that this SCD transcript is highly expressed in adipose tissue and liver, and in the mammary gland of lactating animals. A lower level of expression was detectable in a variety of other tissues including brain. Levels of the SCD transcript were decreased in adipose tissue during lactation, and this appears to be related to a marked decline in serum insulin and insulin-responsiveness of the tissue. Southern analysis of ovine and mouse genomic DNA demonstrated that the ovine SCD cDNA hybridised in a manner consistent with a single gene for SCD in ovine DNA; mouse genomic DNA produced a pattern of hybridisation consistent with the previously characterised mouse SCD-1 and SCD-2 genes. Three ovine cosmids were isolated that comprised the restriction fragments predicted by the genomic Southern analysis. The ovine SCD gene was predicted to be encompassed within a 23 kbp region that was present in all three cosmids. These results demonstrate that SCD is transcribed from a single gene in the ovine genome and this gene is insulin-responsive in ovine adipose tissue. q 1998 Elsevier Science B.V. Keywords: Stearoyl-CoA desaturase; cDNA; Gene; Adipose tissue; Lactation
1. Introduction Abbreviations: ACC, acetyl-CoA carboxylase; cDNA, complementary deoxyribonucleic acid; kbŽp., kilobase Žpairs.; MMLV, murine moloney leukemia virus; ORF, open reading frame; RNase, ribonuclease; SCD, stearoyl-CoA desaturase; UTR, untranslated region ) C orresponding author. Fax: q 44-1292-674003; E-mail: [email protected] 1 The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AJ001048.
Stearoyl-CoA desaturase ŽSCD. catalyses the oxidation of palmitoyl-CoA and stearoyl-CoA at the D 9 position to form the monounsaturated fatty acyl-CoA esters, palmitoeyl-CoA and oleoyl-CoA, respectively; stearoyl-CoA is the preferred substrate w1x. In rodents, SCD enzyme activity is the result of transcription from two related genes Ž SCD-1 and SCD-2. in a
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 2 1 0 - 5
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tissue-specific fashion, each of which encode a highly similar functional enzyme w2,3x. SCD-1 is mainly expressed in adipose tissue and liver of rodents; SCD-2 is principally expressed in the brain w2,3x. The importance of SCD is indicated by the fact that the proportion of saturated and monounsaturated fatty acids of a cell are reflected, not only in the composition of the triacylglycerol, but also in the membrane phospholipid. The ratio of stearic acid to oleic acid incorporated into phospholipids has a significant influence on membrane fluidity, and alteration in this ratio has been implicated in a variety of disease states such as cancer, diabetes, obesity, vascular and coronary heart disease w4–9x. With respect to vascular and coronary disease, a major risk factor for progression is the consumption of high levels of saturated fat in the diet w10x, principally from the meat of domestic ruminants. Ruminant animals in general, and sheep in particular have relatively high levels of saturated to monounsaturated fatty acids in their lipids w11x. This arises from a variety of factors. Dietary fatty acids Žwhich are usually mostly polyunsaturated. are hydrogenated by rumen microorganisms; hence, a large proportion of the dietary fatty acids absorbed are saturated w11x. Also, as ruminant diets have a relatively low fat content, there is a high rate of fatty acid synthesis in adipose tissue, the principal products of which are palmitoyl-CoA and stearoyl-CoA. Ruminant tissues have SCD activity but little is known about its regulation. As increasing the degree of desaturation of the lipid of ruminant tissues, especially adipose tissue and milk triacylglycerol, is likely to be beneficial from the point of view of human nutrition, we have investigated the SCD system of ovine tissues.
2. Materials and methods 2.1. Animals and tissue culture Sheep were Finn–Dorset crossbred animals, fed on hay and cereals for at least four weeks before slaughter w12x. Animals were used either as control Ž nonpregnant non-lactating., pregnant or lactating ewes or wethers Žsix month-old castrated-males.. Female sheep were all 3–5 years old and multiparous. Pregnant animals were used between 100–105 days of
gestation and lactating ewes, suckling at least two lambs, were used at about day 18 of lactation. Animals were anaesthetised, exsanguinated and samples of subcutaneous adipose tissue obtained, snap frozen and stored in liquid nitrogen. Subcutaneous adipose tissue removed aseptically from lactating animals was used to prepare explants. These were maintained in culture in Medium 199 w12x for periods of up to 72 h in the presence or absence of insulin Ž 17 nM. plus dexamethasone Ž 10 nM.. Fresh explants of adipose tissue and explants after culture were snap-frozen and stored in liquid nitrogen. 2.2. Isolation of cDNA clones An ovine adipose cDNA library was constructed in lgt-11 using cDNA prepared from reverse-transcription of adipose tissue RNA w13x by MMLV reverse transcriptase using oligo-dT as a primer. The cDNA was double-stranded and then ligated to EcoRI–NotI adaptors Ž Pharmacia Biotech. prior to ligation into EcoRI restricted and dephosphorylated lgt-11 vector by standard methods w14x. The library was plated on Y1089 host bacteria and replica-plated onto nitrocellulose filters. The filters were hybridised with a rat SCD-1 cDNA corresponding to the coding region of the mRNA w15x radioactively labelled to high-specific activity by the random-priming method w16x. After several rounds of screening, positive recombinant plaques were purified to homogeneity, and the resulting bacteriophage DNA was isolated by standard methods w17x. 2.3. Isolation of genomic clones High molecular weight genomic DNA was isolated from sheep spleen by standard methods w18x. The DNA was partially digested with Sau3AI restriction endonuclease and a fraction corresponding to a size of 33 to 50 kbp was isolated by sucrose-density gradient centrifugation w19x. The resulting DNA was then ligated into the SuperCos I cosmid vector ŽStratagene. and packaged using Gigapack II gold extracts ŽStratagene.. The library corresponding to 8 = 10 5 individual recombinants was plated on the XL1-blue MR host. Aliquots of the library were replica-plated and screened with a radioactively labelled w16x ovine SCD cDNA Ž clone 3A, see below. .
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Cosmids were purified to homogeneity by several rounds of screening. Cosmid DNA was isolated by standard plasmid protocols. Southern analysis of genomic DNA and cosmids was performed using standard methods w14x; filters were washed at 0.2 = SSCr0.1% Žwrv. sodium dodecyl sulfate at 558C Ž20 = SSC s 3 M NaCl, 0.3 M sodium citrate. . 2.4. DNA sequencing and computer analysis Inserts from lgt-11 recombinant bacteriophage were excised with NotI and subcloned in pGEM ŽPromega. plasmid vectors. DNA sequencing was performed by the dideoxy-chain termination method w20x using w a- 35 SxdATP and Taq DNA polymerase with M13rpUC forward and reverse sequencing primers or gene-specific primers ŽCustom-synthesised by Cruachem, Glasgow. . DNA sequence was analysed for similarity using the GCG package Ž University of Madison, WI.. 2.5. RNase protection assay An RNase protection assay was developed using a fragment of the ovine SCD cDNA as a probe. A 392 nucleotide NcoI–EcoRV fragment of clone 1D was subcloned into pGEM-7zfq and used to generate an antisense transcript using SP6 RNA polymerase and a- 32 P CTP w21x. Tissue samples were powdered in a mortar and pestle using liquid nitrogen and homogenised in 5 M guanidinium isothiocyanate, 100 mM EDTA, pH 7.0, using a constant tissue to volume ratio. Aliquots Ž40 m l. of these were hybridised to the antisense transcript and subsequently digested using RNaseArRNase T1 and proteinase K w22x. In addition, various amounts of sense transcript were hybridised to the antisense transcript as molar standards to allow SCD transcript abundance to be determined. After extraction with phenol and chloroform, the samples were precipitated twice and rinsed with 80% Žvrv. alcohol. Samples were dried, resuspended, and after denaturing in formamide loading buffer at 858C, resolved on a 6% Ž wrv. acrylamider8 M urea sequencing gel w23x. After drying, the gels were exposed to a Kodak phosphor screen overnight. The resulting images were scanned using a Molecular Dynamics phosphorimager 445 SI and the volume of individual bands obtained using ImageQuant software. Homogenate DNA content was assayed by the
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method of Labarca and Paigen w24x after extraction of the same tissue homogenate with four volumes of water-saturated chloroform. SCD mRNA was expressed as transcripts per cell by dividing the number of transcripts per volume of homogenate by the cell number per volume of homogenate Žobtained by dividing the DNA content in pg by the diploid DNA content of a cell, assuming a cell contains 7 pg of genomic DNA w25x.. 2.6. Preparation of adipocytes and stromoÕascular cells Adipocytes were prepared by collagenase digestion of omental adipose tissue, essentially as described previously w26x. Adipocytes were recovered by flotation, after which the stromovascular cells in the infranatant were pelleted by centrifugation at 4200 = g for 10 min. The adipocytes and stromovascular cells were resuspended in 5 M guanidine isothiocyanate, 100 mM EDTA pH 7.0, and portions of the cell homogenates were used in the SCD mRNA RNase protection assay and for DNA determination as described above. 2.7. Statistical analysis Data was analysed by one-way ANOVA. 3. Results 3.1. Isolation of cDNA clones from the oÕine omental adipose tissue library For the initial screening of the cDNA library 2 = 10 5 recombinant plaques were screened with a 1.2kbp cDNA insert corresponding to rat SCD-1 w15x. The primary screen resulted in 12 positives, of which nine were purified to homogeneity after successive rounds of screening. The cDNA inserts from the l recombinants were subsequently sub-cloned into plasmid pGEM-7zfŽq. and initially characterised by restriction endonuclease mapping and DNA sequencing ŽFig. 1.. Restriction endonuclease mapping of the recombinants essentially described one species of cDNA that was subsequently confirmed by complete or partial DNA sequencing of the nine recombinants. The sequence data from all the recombinants indi-
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Fig. 1. Strategy for cloning and sequencing ovine SCD cDNAs. An oligo-dT sheep adipose cDNA library was screened with a rat SCD-1 cDNA insert w15x. The resulting ovine clones are shown with reference to the structure of the rat SCD-1 mRNA. The shaded box denotes the open-reading frame of rat SCD-1. Restriction enzyme abbreviations ŽA s ApaI; E s EcoRV; H s HindIII; Nc s NcoI; Ns s NsiI; P s PstI; S s SacI; Xa s Xba; Xo s XhoI.. The black arrowheads and dotted lines show the direction and extent of individual sequencing reactions. The black box denotes the 392 nucleotide Nc–E fragment used in the RNase protection assay ŽFigs. 4 and 8..
cated only one open reading frame Ž ORF. for the SCD mRNA species, principally expressed in ovine adipose tissue. The complete coding sequence contains 1077 nucleotides encoding a protein of 359 amino acids with a calculated Mr of 41 742 ŽFig. 2.. The largest recombinant Ž3D. to contain a complete ORF approximated to 3.4 kbp which is significantly smaller than the estimated size of 5 kb from Northern blotting of ovine adipose RNA Žresults not shown.. Furthermore, this recombinant did not possess a polyA tract nor a polyadenylation motif at its 3X terminius despite being isolated from an oligo-dT primed cDNA library. This suggests that this clone and the other SCD recombinants isolated from the cDNA library resulted from non-poly-A tail priming of mRNA. In rodents, SCD activity is the result of transcription from two related genes ŽSCD-1 and -2. in a tissue-specific fashion. Complementary DNAs corre-
sponding to the complete ORF for rat w15x and mouse SCD-1 w2x and mouse SCD-2 w3x have been previously described; partial sequences have also been derived for rat SCD-2 w28x and human SCD w29x. In addition cDNAs related to these SCDs have been isolated for the sheep tick Amblyomma w30x, the protozoan Tetrahymena w31x and yeast w32x. Comparison of these amino acid sequences for the higher eukaryotes Žusing the GCG Homologies algorithm. demonstrate that the ovine SCD amino acid sequence has 89–93% identity with mouse, rat and human SCDs, with a somewhat lower identity with Amblyomma SCD Ž74%. and the other lower eukaryotes Žresults not shown.. However, it is not possible to ascertain whether the ovine SCD has greater identity with mouse SCD-1 or with mouse SCD-2, as both appear to diverge approximately equally from the ovine SCD Ž89% identity. .
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Fig. 2. The nucleotide sequence of ovine SCD cDNA. The codon for the initiator methionine and the stop codon are underlined. The open-reading frame is denoted by three letter abbreviations for the amino acids. The His residues indicated by bold script correspond to eight histidines spatially conserved in the family of membrane desaturases which are thought to act as iron-centres in the active site of SCD w27x.
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Sequence comparisons of the 5X UTRs of the SCDs demonstrates that the ovine SCD 5X UTR exhibits a higher identity with rat and mouse SCD-1 Žboth 64%. as opposed to the corresponding SCD-2s Žboth 58%.. The similarity between the ovine SCD and the rodent SCDs is particularly marked around the translation initiation codon and then subsequently diverges towards the 5X termini of the respective SCD cDNAs ŽFig. 3. . Conversely, comparison of 695 nucleotides downstream of the termination codon Ž clone 1D, Fig. 1. in the ovine SCD mRNA 3X UTR with the corresponding rodent SCD cDNAs demonstrates a
55% identity with mouse SCD-2, which is significantly higher than the identity with rat and mouse SCD-1s Žboth 47%.. Such comparison is interesting, as similar analysis reveals a low identity in the 3X UTR between mouse SCD-1 and mouse SCD-2 Ž 44%. and a relatively high identity between mouse and rat SCD-1 Ž81%.. 3.2. Expression of SCD gene in oÕine tissues To determine the relative expression of the SCD transcript in a variety of ovine tissues, an RNase
Fig. 3. Multiple alignment of the 5X untranslated region of ovine Žov. SCD cDNA with those of mouse Žm. and rat Žr. SCD-1 and SCD-2 cDNAs. The alignment was performed with the CLUSTALV program. Black box areas denote sequence conservation. Numbers at the right hand margin indicate the nucleotide residues.
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protection assay using a 392 nucleotide NcoI–EcoRV fragment of clone 1D ŽFig. 1. was established. An RNase protection assay also has the advantage of being able to evaluate the number of copies of the SCD transcript in the ‘average’ cell comprising the tissue of interest, assuming a cell contains approximately 7 pg of genomic DNA w25x. The results of such analysis is shown in Fig. 4A,B. This demonstrates that although the SCD transcript was detected in all tissues examined from wether sheep, the highest levels of expression per volume of tissue homogenate appeared to be in liver and in the mammary gland of lactating animals Ž Fig. 4A. . However, when the DNA content of the homogenate was taken into account and the results expressed as SCD transcripts per cell, it can be seen that the highest level of expression, approximating to 100–250 transcripts per cell, was observed in adipose tissue and liver ŽFig. 4B., tissues that are major sites of triacylglycerol synthesis. The apparent discrepancy between the relatively low level of SCD expression in the adipose tissue homogenate Ž Fig. 4A. , and the markedly higher expression when the data is expressed as transcriptsrcell ŽFig. 4B. , is due to adipose tissue homogenates having a low DNA concentration, due in part to approximately 70% of their tissue volume comprising lipid. Fractionation of adipose tissue into the stromovascular cells Žendothelial cells and fibroblasts. and an adipocyte-enriched fraction demonstrated that expression of SCD mRNA in the adipocyte fraction was 140-fold higher per cell than in the stromovascular fraction Žresults not shown.. This implies that adipocytes, which comprise only 5–10% of the cells per unit tissue w33x, are the principal site of SCD gene expression in adipose tissue. Of the remaining tissues, the range of expression varied from 1 transcript per cell in pancreas to 31.5 transcripts per cell in muscle; SCD mRNA was readily detected in ovine brain with approximately 10 transcripts per cell. Additionally, high levels of SCD mRNA were also found in the mammary gland from lactating sheep ŽFig. 4B. . 3.3. OÕine SCD is transcribed from a single gene in the oÕine genome Southern analysis of ovine genomic DNA restricted with a variety of restriction endonucleases
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Fig. 4. Determination of SCD mRNA in ovine tissues by RNase protection assay. ŽA. Homogenates were prepared from various ovine tissues and these were used in a RNase protection assay with antisense SCD probe resulting in a 392 nucleotide protected fragment. The band of 467 nucleotides is undigested antisense SCD probe and represents approximately 0.1% of the probe added to each assay. ŽB. The 392 nucleotide protected fragments were quantitated using a phosphorimager with ImageQuant software. DNA content of the homogenates was determined and SCD mRNA was expressed as the number of transcriptsrcell by dividing the number of transcripts per volume of homogenate by the cell number per volume homogenate Žobtained by dividing the homogenate DNA content in pg by the diploid DNA content of a cell, assuming a cell contains 7 pg of genomic DNA w25x.: Br s Brain; K s Kidney; H s Heart; M s Muscle; P s Pancreas; Lu s Lung; L s Liver; SpsSpleen; Ad s Adipose tissue; LMG s lactating mammary gland. Values are means"S.E.M. from three animals.
and then hybridised with a SCD cDNA corresponding to the ORF Žclone 3A. generated a relatively simple hybridisation pattern Ž Fig. 5A. ; a variety of
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Fig. 5. Southern analysis of genomic DNA isolated from ŽA. sheep and ŽB. mouse and then hybridised with ovine SCD cDNA. Twenty m g of genomic DNA was digested with restriction enzymes as indicated and then separated on a 0.8% Žwrv. agarose gel, which was blotted onto a nylon membrane. This was hybridised with ovine SCD cDNA insert Žclone 3A.. Numbers at the left hand margin in ŽA. indicate the position of DNA markers. Restriction fragments in the mouse genomic DNA ŽB. that hybridised with ovine SCD cDNA are indicated with reference to fragments that have been reported to hybridise with the homologous murine SCD-1 cDNA w2x and that have been identified in the murine SCD-1 w2x and SCD-2 w3x genes.
enzymes Ž EcoRI, BamHI, SacI. principally generated a single large hybridisation fragment Ž approximately 21 kbp in the case of EcoRI. whereas others Ž HindIII, Pst I. generated smaller multiple fragments. A HindIII site is present in the SCD ORF Ž corresponding to murine SCD exon 2. , and thus accounts for the two fragments generated by HindIII of 5 kbp and 8 kbp in Fig. 5A. By comparison, Southern analysis of mouse genomic DNA digested with EcoRI ŽFig. 5B. and then hybridised with the ovine SCD cDNA generates a more complex pattern of hybridisation; fragments of 14.0, 9.0, 4.5 and 2.2 kbp were generated. Fragments of similar size can be observed
in the murine SCD-1 and SCD-2 genes w2,3x. A similarly complex pattern of hybridisation was observed with mouse DNA digested with HindIII; fragments of similar size can be observed in the murine SCD-1 and SCD-2 genes w2,3x, although a complete restriction map for HindIII is not available for the mouse SCD-2 gene w3x. Screening of an ovine cosmid library Ž 4 = 10 5 colonies. with the SCD cDNA corresponding to the ORF Žclone 3A. resulted in the isolation of three clones Žcosmids 3, 7 and 8. . These cosmids were restriction-mapped and were demonstrated to correspond to an overlapping series of genomic DNA
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clones ŽFig. 6.. Ovine SCD cDNA clone 3A hybridised to a 21-kbp EcoRI fragment in all the cosmids. Additionally, each cosmid hybridised to SCD cDNAs corresponding to both the 5X UTR and the terminal portion of the 3X UTR, suggesting that the entire SCD transcriptional unit was represented within a genomic region that approximated to 23 kbp. This comprised the 21 kbp EcoRI fragment and a 2-kbp EcoRI fragment that hybridised with the 3X UTR cDNA ŽFig. 6.. The ends of each cosmid were distinct and comprised multiple EcoRI fragment repeats of 0.8 kbp. Furthermore, double-stranded DNA sequencing of each of the three cosmids using an oligonucleotide predicted to occur within exon 2 Žby analogy with the structures of the two murine genes. as a primer generated the same nucleotide sequence; the 3X portion of exon 2 was conserved as predicted and the sequence of the 5X region of the adjacent intron was identical in the three cosmid clones Ž results not shown.. Digestion of the cosmids with a variety of restriction enzymes used in the Southern analysis of ovine genomic DNA Ž Fig. 5A. generated essentially the same pattern of hybridisation ŽFig. 7. . For example, digestion of each of the cosmids with HindIII generated the 5.0 and 8.0 kbp fragments that hybridised with clone 3A; digestion with Pst I produced fragments of 4.0, 2.5 and 0.7 kbp that hybridised with clone 3A and that were also present in the genomic DNA Ž Fig. 5A. . These results demonstrated that a single genomic unit corresponding to the ovine SCD gene had been isolated from the
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Fig. 7. Southern analysis of cosmid 3 demonstrating that restriction fragments in ovine genomic DNA that hybridised to SCD cDNA are present in the cosmid clone. One m g of cosmid DNA was digested with the complement of restriction enzymes used in Fig. 5A and Southern-blotted. This was hybridised with ovine SCD cDNA Žclone 3A.. Numbers at the left hand margin indicate the position of DNA markers.
cosmid library with a structure predicted by the Southern blotting of the restricted genomic DNA. 3.4. The SCD gene is repressed in adipose tissue during pregnancy and lactation Lactation results in a major decrease in the expression of the SCD gene in sub-cutaneous adipose tissue, being reduced from 678 " 271 mRNA copies per cell in non-pregnant non-lactating animals to 76 " 35 mRNA copies per cell during lactation Ž P - 0.05. ŽFig. 8. . A similar level of SCD gene repression was
Fig. 6. Restriction fragments corresponding to the ovine SCD gene are encompassed within three clones of the ovine cosmid library. Cosmids 3, 7 and 8 were digested with various restriction enzymes ŽEI s EcoRI; H s HindIII; S s SacI. and Southern blotted. The blot was sequentially hybridised with cDNA inserts corresponding to the coding region Žclone 3A., the 5X UTRrN-terminal coding region Žan ApaI–ApaI subclone of 3D. and the 3X UTR Ža NsiI–NcoI subclone of 3D.. The position of the fragments that hybridised with each of the cDNAs are indicated by the shaded boxes. Each of the cosmids comprised a 21-kb and a 2-kb EcoRI fragment that hybridised with one or more of the ovine SCD cDNAs. The ends of each cosmid were distinct and comprised EcoRI fragment repetitive elements that are not shown.
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4. Discussion
Fig. 8. Expression of SCD mRNA in adipose tissue during pregnancy and lactation and in explants from lactating sheep cultured with insulin plus dexamethasone. Homogenates were prepared from subcutaneous adipose tissue from non-pregnant non-lactating ŽC., 100–105 day pregnant ŽP. and lactating animals ŽL., and from sub-cutaneous adipose explants from lactating sheep at time zero Ž0. and after culture for 72 h with no hormones ŽNA. or with insulin plus dexamethasone ŽIqD.. These were used in an RNase protection assay with antisense SCD transcript resulting in a 392 nucleotide protected fragment. The protected fragments were quantitated using a phosphorimager with ImageQuant software. DNA content of the homogenates was determined and SCD mRNA was expressed as the number of transcriptsrcell, assuming a cell contains 7 pg of genomic DNA w25x. Values were analysed by ANOVA and are means"S.E.M. of seven animals; ) ) ) P - 0.001 compared with explants at zero time.
also evident in the sub-cutaneous adipose tissue depot of animals that were 100 days pregnant ŽFig. 8.. Preparation and culture of adipose tissue explants from the sub-cutaneous depot of lactating sheep in Medium 199 containing insulin plus dexamethasone for 72 h results in an approximate 15-fold increase in the level of SCD transcripts from 151 " 37 mRNA copies per cell in the freshly prepared explants to 2198 " 340 mRNA copies per cell at the end of culture with the added hormones Ž P - 0.001., approximately 3-fold higher than the level of SCD expression in the sub-cutaneous depot of non-pregnant non-lactating sheep ŽFig. 8. . Culture of adipose explants for 72 h in Medium 199 without added hormones resulted in a level of SCD gene expression similar to the freshly prepared explants.
In mice, SCD mRNA is transcribed from two evolutionary related genes, SCD-1 and SCD-2 w2,3x in a tissue-specific fashion. For example, SCD-1 is the major transcript in adipose tissue and liver w3x. SCD-1 does not appear to be expressed in brain; SCD transcripts in brain are derived from the SCD-2 gene. Conversely, the SCD-2 gene is not transcribed in liver and is only transcribed at low levels in adipose tissue w3x. In this present study, we have isolated nine independent clones corresponding to ovine SCD from an adipose tissue cDNA library; these clones correspond to the same molecular species as judged by restriction mapping and DNA sequence. In contrast to mouse SCD mRNA, this ovine adipose tissue SCD transcript appears to be widely expressed, though expression is highest in adipose tissue and liver and in the mammary gland of lactating animals. This SCD transcript is readily detectable in ovine brain. These differences in the expression profile of SCD transcripts in mice and sheep are unlikely to be attributed to assay sensitivity, as both ourselves and Kaestner et al. w3x used RNase protection assays to evaluate SCD gene expression. Southern analysis of ovine genomic DNA using a cDNA encompassing the SCD ORF demonstrated a relatively simple pattern of fragment hybridisation; a number of restriction enzymes generated a single hybridisation fragment. This contrasts with mouse genomic DNA in which mouse SCD-1 cDNA recognises multiple bands in the mouse genome even at high stringency, some of which can be accounted for by the mouse SCD-2 gene. Indeed, the SCD-2 gene was isolated from mouse genomic DNA libraries using the mouse SCD-1 cDNA w3x. Interestingly, ovine SCD cDNA hybridises with multiple fragments generated by a number of restriction endonucleases in the mouse genome; on the basis of size, these can be assigned to either the SCD-1 or the SCD-2 genes w2,3x. Thus, the relatively simple hybridisation pattern observed with ovine genomic DNA is consistent with there being a single copy of the SCD gene in the haploid ovine genome. Indeed, screening of an ovine cosmid library with SCD cDNA generated an overlapping series of clones corresponding to a single genomic region; this was confirmed by DNA sequencing of the region predicted to represent the
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boundary of the 2nd exon and the 2nd intron, and the presence of restriction fragments occurring within the cosmid clones that were predicted to occur in the SCD locus from the genomic Southern blots. Thus, it appears that the gene duplication that resulted in the SCD-1 and SCD-2 genes in mouse w2,3x and rat w15,28x is a relatively recent event in evolution, occurring after the species divergence of rodents and ruminants. It is not clear why rodents have evolved two SCD genes and sheep only one, although it could relate to the differing complexity of the tissue-specific control of lipid metabolism in each species. Rodents, in the wild at least, exhibit marked diurnal variations in food intake and have diets of variable fat content with the result that different tissues would have different temporal requirements for monounsaturated fatty acid; for example, brain would still have a need for monounsaturated fatty acids for myelin formation at times when fatty acid synthesis in adipose tissue is low w34x. Sheep tissues, on the other hand, tend to have a more constant supply of nutrients that are usually low in fatty acids, due to the nature of the diet and the buffering action of the rumen w35x. Lactation results in a profound change in the lipid synthetic activity of adipose tissue in sheep with a major decrease in the rate of fatty acid synthesis, and the activity of the key lipogenic enzyme acetyl-CoA carboxylase ŽACC.; there are also smaller decreases in the rate of fatty acid esterification and the activity of glycerol-3-phosphate acyltransferase and lipoprotein lipase w12,33,36,37x. The decrease in ACC activity is at least partly due to a fall in the amount of ACC mRNA w33x. The present study shows that there is also a fall in SCD mRNA with lactation. The changes in lipid synthetic activity in sheep adipose tissue as described above occurs around day 105 of pregnancy w33,36x at a time when demands for nutrients of the growing foetusŽes. and mammary glands are rapidly increasing. The amount of SCD mRNA is also decreased by day 105 of pregnancy. The major factors responsible for the decreased capacity for lipid synthesis in adipose tissue during late-pregnancy and during lactation are thought to include insulin and growth hormone, the serum concentrations of which decrease and increase respectively w36x, and insulin resistance of adipose tissue w37x. Maintenance of explants of adipose tissue from lactating sheep in culture for 72 h with insulin plus
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dexamethasone restores the rate of lipogenesis to levels found in non-lactating ewes, and increases the amount of ACC mRNA to levels in excess of those found in non-lactating ewes w33x; culture with insulin plus dexamethasone had a similar effect on the amount of SCD mRNA. This enhanced level of expression obtained during culture with insulin plus dexamethasone may be explained by a change in the balance of activators and repressors of the SCD and ACC genes resulting in a greater relief of repression in the in vitro system. This is likely to be achieved by the absence of negatively acting hormones, such as growth hormone that has been implicated in repressing ACC and fatty acid synthase gene expression in adipose tissue of pigs and rats, respectively w38–40x. The mechanism for the repression of the SCD gene, and how this is coordinated with suppression of lipogenesis is unknown, though as both parameters are relieved by insulin plus dexamethasone in vitro, suggests that attenuation of a common or related signalling event may be involved. Future consideration of the mechanism for the repression of the SCD gene in ovine adipose tissue during lactation and its subsequent de-repression in vitro will require characterisation of the promoter to elucidate the factors that regulate transcription of the gene. Recently, 4.3 kb of the mouse SCD-1 5X flanking sequence has been shown to be insulin-responsive in transient transfection of a mouse liver cell line, suggesting the presence of an insulin-response element within the transfected DNA w41x. DNA sequence comparison of the 5X flanking sequence of the ovine SCD gene with that of the mouse SCD-1 gene may aid in the identification of regulatory domains within each promoter.
5. Conclusion The activity of SCD in the adipose tissue of sheep and other ruminant animals is likely to be critical in determining the saturated fat content of meat. Any attempt at altering the fatty acid composition in favour of less saturated fatty acids may be dependent on manipulation of the expression of the SCD gene. Our findings of only a single gene in the ovine genome and that expression of the gene is under hormonal control may help in designing such strategies.
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Acknowledgements The authors thank Mrs. Mandy Vallance and Mrs. Helen Pollock for expert technical assistance. This work was funded by a Link grant from the Biotechnology and Biological Sciences Research Council and the Scottish Office Agriculture Environment and Fisheries Department. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AJ001048.
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