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A new elongase selectively expressed in Drosophila male reproductive system
BBRC Biochemical and Biophysical Research Communications 333 (2005) 1066–1072 www.elsevier.com/locate/ybbrc
A new elongase selectively expressed in Drosophila male reproductive system Thomas Chertemps, Line Duportets, Carole Labeur, Claude Wicker-Thomas * Laboratoire de Neurobiologie de lÕApprentissage, de la Me´moire et de la Communication, CNRS UMR 8620, Baˆt. 446, Universite´ Paris-Sud, 91405 ORSAY Ce´dex, France Received 24 May 2005 Available online 13 June 2005
Abstract We have identified an elongase gene, elo68a, which is specifically transcribed in males. We have characterized the elo68a open reading frame, expressed it in fasD elo1D yeast and showed that it could elongate myristoleic and palmitoleic acids, therefore sharing an Elo1 specificity. This elongase was found to be exclusively expressed in male genital system (testis and ejaculatory bulb). Northern blot analysis showed that the elo68a gene was inducible at low temperatures. One P-strain mutant for elo68a and three excision lines for this P-element were subsequently studied. The excision line with only 1% elo68a expression showed decreased levels of vaccenyl acetate, a male pheromone produced in the ejaculatory bulb. The induction of elo68a expression at 21 C was also paralleled with higher vaccenyl acetate production. These results strongly suggest that elo68a is involved in the elongation of short unsaturated fatty acids in males and might play a role in vaccenyl acetate biosynthesis. 2005 Elsevier Inc. All rights reserved. Keywords: Elongase; Drosophila melanogaster; Fatty acid; Elo1; Vaccenyl acetate
In Drosophila, palmitoleic and oleic acids represent half of the total fatty acids [1]. They serve not only as membrane structural components but are also used for energy storage and as bioactive metabolites. Moreover, they are the precursors of sex pheromones, which act during courtship by contact, or at very short distance [2,3]. Pheromone biosynthesis involves a desaturase, Desat1, which introduces a D9 desaturation into palmitic acid [4,5]. Palmitoleic acid is then elongated through elongases, thus leading to vaccenic acid and to long chain x7 monoenic fatty acids which are eventually decarboxylated to give 7-unsaturated hydrocarbons. Vaccenic acid is also the precur*
Corresponding author. Fax: +33 1 69 15 77 26. E-mail address: [email protected] (C. Wicker-Thomas). 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.015
sor of vaccenyl acetate (cVA), a potent nonhydrocarbon male pheromone produced in the ejaculatory bulb and transferred to the female during mating. This compound has been shown both to inhibit male courtship behaviour and to act as an aggregation pheromone [6–8]. In this report, we have characterized an elongase gene (named elo68a) which was specifically expressed in male reproductive system, with a strong expression in testis and a faint expression in ejaculatory bulb. We expressed the elo68a open reading frame in yeast and showed that it encodes an enzyme which can elongate myristoleic and palmitoleic acids. A P-element insertion in the upstream region of elo68a and excision of this transposon were studied. The results suggest that elo68a might be involved in male cVA biosynthesis.
T. Chertemps et al. / Biochemical and Biophysical Research Communications 333 (2005) 1066–1072
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Materials and methods
Results and discussion
Cloning and the expression of Drosophila elo68a in Saccharomyces cerevisiae. 5 0 and 3 0 RACE amplifications were performed to obtain a full-length cDNA sequence, using a 5 0 /3 0 Race kit (Roche Applied Bioscience). The elo68a open reading frame was amplified by RT-PCR from Drosophila adult cDNA using two specific primers (5 0 -CGCGG ATCCCGCATGGCATCACTTGGCATCTCA-3 0 ) and (5 0 -AGCGG TACCTTATATAGGTTTCTTGCCGAC-3 0 ) and cloned directly into a PYEDP vector at BamHI and KpnI sites [9]. The resulting galactoseinducible constructs were introduced into S. cerevisiae strain DTY001 (MATa, fas2::LEU2, elo1::HIS3, leu2-3, leu2-1/2, can1-100, ura-3, ade2-1, his3-11, and his3-15) strain ([10], kindly given by C.E. Martin), using the lithium acetate method. The transformed cells were selected and maintained on uracil-deficient medium: yeast extract/nitrogen/ base (YN) containing 20 lg/ml adenine, 1% tergitol type NP-40, 2% dextrose, and 0.5 mM C14:0. The yeast transformants were cultivated at 30 C, in 50 ml YN containing 20 lg/ml adenine, 1% tergitol, 2% raffinose, and 0.5 mM fatty acid substrate, until the cell density reached 5 · 106 cells/ml. The expression of the transgene was induced by the addition of 2% galactose and additional cultivation for 30 h. Fatty acids were extracted in chloroform/methanol and transformed into methyl esters as previously described. They were then analyzed by GC/MS [5]. Fly stocks and genetics. The control strains, Canton-S and w;TM3 were obtained from the Bloomington Center. The GS11513 line, which harbors a single P-element (GSV6 vector) 502 bp upstream of the elo68a open reading frame, was obtained from the DGSP (Kyoto). To generate excision lines, an imprecise P-element excision strategy was used, as previously described [11]. Drosophila cultures were kept at 25 C in a 12-h light and 12-h dark cycle on standard yeast/cornmeal/agar medium. For experiments, flies were isolated just after emergence and sexes were held separately until use. cVA analysis. cVA of 4-day-old flies was extracted individually in heptane and analyzed by gas liquid chromatography (Perkin Elmer AutoSystem) [5]. Characterization of the excision lines. Three excision lines were characterized at the genomic level. Genomic DNA was amplified by PCR using the primer pair CG32071-5 0 (5 0 -ATGGCATCCAGAATC CGTAGA-3 0 ) and CG32071-3 0 (5 0 -TTAGTAGCGTCTGAAGCGG CG-3 0 ). The locations of these primers are: 867–846 and 435–414 bp upstream of the elo68a open reading frame. Visualisation of the elo68a expression. The elo68a expression in flies was conducted using a vital GFP reporter. The GSV6 vector inserted in the GS11513 line contains basal hsp70 promoter adjacent to a GFP reporter near its 3 0 end. Upon activation of the hsp70 promoter, expression of the labeling GFP gene occurs, which made it possible to visualize the target tissues under the control of 5 0 P adjacent genomic region. The activation of the hsp70 promoter was performed through two series of heat-shock per day (20 min at 37 C, 20 min at 20 C, 20 min at 37 C), from the emergence until 4 days after emergence. Images were visualized and photographed on a Nikon eclipse E800 microscope with a Cool Snap camera. RNA and Northern blot analysis. RNA was extracted from third instar larvae or 4-day-old adults by the Trizol (Gibco) method, according to the procedures recommended by the manufacturer. The total RNA was resolved on a 1% agarose/formaldehyde gel (30 lg/slot) and then transferred to a Positive HCD membrane membrane (Appligene). The blot was prehybridized and hybridized, using the elo68a open reading as a probe [4]. The blots were detected immunochemically with Phototope-Star Chemiluminescent Detection Kit (Biolabs) on Fuji RX film. The film was scanned on a Geldoc 2000 (Bio-Rad) and bands were quantified using the QuantityOne software. Statistics. The data are presented as means ± SEM. An analysis of variance (StudentÕs t test) was used for the statistical analyses, and p < 0.05 was accepted as statistically significant.
Identification of two putative D. melanogaster elongases Two potential elongase genes, designated elo68a (Flybase no. CG32072) and elo68b (Flybase no. CG11801), and another gene with no known homology (Flybase no. CG32071), were identified in the cytological region 68A6 (Fig. 1A). CG32071 and elo68b could not be amplified from either male or female cDNA. elo68a, located 326 nucleotides upstream elo68b, contains three introns and could be amplified by PCR only on male adult cDNA. A mutant line, GS11513, was found to harbor a P-element inserted 502 nucleotides upstream of the initiator codon of CG32072. Structure of the elo68a protein 5 0 and 3 0 RACE amplifications were performed to obtain a full-length cDNA sequence and yielded an open reading frame of 786 nucleotides. The deduced protein, containing 262 amino acids, has 40 more amino acids at the N-terminal end than the protein predicted in Flybase (Fig. 1B). It contains a diiron-oxo binding HXXHH motif in the central part of the protein (residues 140–144). This motif is also present in other fatty acid elongases and is believed to act as Fe-chelating ligand used for the electon transfer for O2-dependent redox reactions [12]. Hydropathy analysis using the Kyte–Doolittle program suggests that it is a transmembrane protein with at least five membrane spanning regions, a characteristic shared by previously identified yeast and mouse elongase enzymes [10,13,14]. Drosophila Elo68a most closely resembles mouse elongase SSC2 (37% identity); the sequence identity of Elo68a to yeast elongases is 29–32% (Fig. 2). Functional expression of elo68a gene in yeast Elongase activities were evaluated in yeast mutant strain DTY001, deficient both in fas2 and elo1 activity. When this mutant was transformed with the empty PYEDP vector and cultivated on medium supplemented with palmitic acid (14:0), significant amounts of 16:0 and 18:0 were present, due to a broad substrate specificity of yeast Elo2 (Fig. 3). Significant levels of 14:1D9, 16:1D9, and 18:1D9 were also generated, due to a D9 desaturation by the yeast endogenous enzyme OLE1. This fatty acid profile did not change after galactose induction of the YEDP vector. To determine whether elo68a encodes a functional enzyme with elongase activity, its open reading frame was cloned into the PYEDP vector (PYEPD-elo68a) and expressed in DTY001yeast (Fig. 3). Without galactose induction, the fatty acid profile did not differ from that obtained with PYEPD alone (with or without induc-
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Fig. 1. The genomic organization of the elo68 locus, the cDNA elo68a sequence and encoded amino acids of the elo68a protein coding sequence. (A) Schematic diagram of the elo68a (CG32072) genomic region. CG11801 and CG32072 encode elongase sequences, but only CG32072 produces detectable transcripts in our analyses. CG32071 is similarly not transcribed. The mutant line (GS11513) contains a P-element inserted 502 nucleotides upstream of the initiator codon of CG32072. (B) The sequences of 961-bp elo68a cDNA and the encoded 262 amino acids are shown. The pair of primer sequences used in the PCR-based cloning is underlined, and the polyadenylation sequence is underlined with a dotted line. The histidine motif is boxed.
tion). After induction of elo68a16:1D11 and 18:1D13 were generated in three subsequent experiments with quantitatively reproducible results, suggesting elongation of endogenous 14:1D9 with 2- and 4-carbon units, respectively. Moreover, a low level of 18:1D11 was observed (1.2%), due to the elongation of 16:1D9. These three unsaturated fatty acids were not detected in yeast transformed with PYEDP alone. If the medium was supplemented with longer chain substrates (18:0, 20:0, 18:1D9 or 18:1D11), no elongation activity could be detected (data not shown). These data suggest that the Elo68a protein has an elongase activity for short chain fatty acids. As far as substrate specificity is concerned, elo68a is more similar to yeast Elo1 as it extends C14 and C16 fatty acids to C16-C18 [10]. Whereas Elo1 is restricted to the elongation of short fatty acids, Elo2 elongates palmitoyl-CoA and stearoyl-CoA up to C22 fatty acids, and Elo3 fatty acids of 20–26 carbons [13]. Animal orthologues acting on fatty acids with chains shorter than C18 have been identified [15–17]. At the protein level,
these mammalian elongases show only 27% identity with Drosophila Elo68a. elo68a expression in wild-type flies Northern blot analysis of elo68a demonstrated specific expression in male adults (Fig. 4A). No detectable signal was obtained in third instar larvae or in female adults; also, no amplification of elo68a could be obtained in female adults and larvae by RT-PCR. This result indicates that elo68a is sex-specifically transcribed. GFP expression under the control of elo68a promoter showed specific labeling in males; this was exclusively found in the reproductive system, with a strong expression in testis, and a faint expression in ejaculatory bulb (Fig. 4B). We then tested the influence of temperature on elo68a expression and found higher expression at 21 C. There was a marked increase in the transcript levels at 21 C (1.5 times more, determined from the Northern blot), compared to 25 and 29 C.
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Fig. 2. Alignment of D. melanogaster Elo68a with yeast (Elo1, Elo2, and Elo3) and mouse (SSC1, SSC2, Cig30, Elovl4, and LCE) elongases. Identical amino acids between two and six sequences are highlighted in light gray, and between seven or nine sequences in dark grey. The conserved histidine motif is boxed. Elo68a is 32%, 31%, 29%, 34%, 37%, 27%, 35%, and 27% identical to Elo1, Elo2, Elo3, SSC1, SSC2, Cig30, Elovl4, and LCE, respectively.
elo68a expression in GS11513 and excised lines The elo68a expression was somewhat increased in GS11513 flies. After excision of the P-element, 60 lines were obtained. Three excision lines were selected for further analysis. The sequences adjacent to the P insertion site in these three excision lines were determined (Fig. 5).
Sequence in ex28 was the same as in wild-type, showing that the excision was precise. In contrast, the P excision in the ex20 and ex25 lines left 44 and 45 nucleotides inserted at the point of the excision, respectively. These insertions contained several nucleotides from the 5 0 and 3 0 end of the P-element and a repeat of eight nucleotides from adjacent genomic sequence.
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Fig. 3. Fatty acid profiles of DTY001 yeast (fasD; elo1D) transformed with an empty PYEDP vector (control) or PYEDPelo68a. Analysis of fatty acid methyl esters by GC-MS with or without induction by the addition of galactose and C14:0 supplementation.
Fig. 4. Expression of elo68a. (A) Northern blot of total RNA from Canton-S strain. RNA was isolated from whole third instar larvae (lane 1) or 4day-old adults reared at 25 C until emergence and then kept at 21 C (lanes 2 and 3), 25 C (lanes 4 and 5) or 29 C (lanes 6 and 7). Thirty micrograms was loaded into each lane of a 1% formaldehyde gel, blotted, and probed with elo68a orf. (B) elo68a expression in male reproductive system. GS11513 males were heatshocked twice a day to induce GFP expression under the control of elo68a promoter and reproductive system dissected at 4 days after emergence. A photograph (400·) of the reproductive system is shown (left); GFP labeling occurs in testis (middle); and ejaculatory bulb (right).
We then investigated elo68a expression in the excision lines and found that elo68a expression was not affected in ex28, but highly reduced in ex25 and ex20, compared to wild-type (Fig. 5 and Table 1).
Vaccenyl acetate and hydrocarbons in GS11513 and excised lines The hydrocarbons of GS11513 and excised flies were affected only in males: a significant increase in 7-trico-
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Fig. 5. Sequences adjacent to the P insertion site in the three excision lines, ex28, ex20 and ex25. The site of insertion at 502 nucleotides (numbered from the first ATG of elo68 open reading frame) is in bold. The sequence found in ex28 is wild-type. The insertion sequences in ex20 and ex25 are underlined. The transcription levels of elo68 were determined from two independent northern experiments and expressed as a percentage of elo68 transcription levels in a w; TM3 wild-type strain.
Table 1 Expression level of elo68 and cVA (ng/male) in flies from control, P or excision lines reared at 25 C (A) and in Canton-S flies maintained at different temperatures from emergence (B) (A) Line
w; TM3
ex20
ex25
ex28
P/TM3
P/P
Expression cVA
100 204 ± 21 a
1 130 ± 32 b
26 259 ± 73 a
80 275 ± 60 a
100 270 ± 41 a
116 226 ± 37 a
(B) Temperature (C)
21
25
29
Expression cVA
150 316 ± 38 b
100 271 ± 15 a
100 286 ± 34 a
Values indicate means ± SEM of 10 independent measurements. In each line, values followed by the same letters are not significantly different at the 0.05 probability level.
sene was observed in GS11513 line and in several excision lines (data not shown). However, this phenotype could not be directly linked to the level of expression of elo68a. As cVA is exclusively produced in male ejaculatory bulb, it was quantified in excision lines and in control flies maintained at different temperatures (Table 1). cVA production was reduced by half in ex20, which shows only 1% elo68a expression. It was not significantly different in the other lines, which were kept at 25 C. However, cVA levels significantly increased in control flies maintained at 21 C, in which elo68a expression was higher. These results suggest that Elo68a might be involved in cVA biosynthesis.
Conclusion This report is the first characterization of an elongase gene in Drosophila. We have found 20 elongase sequences in the Drosophila genome, with homology to yeast and mouse elongases. Several elongases might share similar substrate specificity and the effect on a mutation of one elongase might be partially compensated by the activity of another gene. Nonetheless, we found a mutant phenotype associated with the inactivation of elo68a in male flies: a 50% decrease in levels of vaccenyl acetate occurred when elo68a expression was reduced to 1% of the wild-type level. Moreover, at a relatively lower temperature, elo68a expression is enhanced and an increased production of cVA occurs. As cVA is exclusively
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produced in the ejaculatory bulb, where elo68a is also expressed, it seems likely that elo68a is involved, at least partially, in vaccenyl acetate biosynthesis and could therefore control the elongation step of palmitic acid to vaccenic acid, as the in vitro assay suggests. On the other hand, the reproductive system is not involved in hydrocarbon production. The modification of 7-tricosene in some lines might be an indirect consequence of elo68a activity and/or of other elongases. No significant effect on drosophila total fatty acids was observed either (data not shown), and may be due to the very specific elo68a expression in the male reproductive system. We are continuing to characterize other Drosophila elongases and ultimately, these advances should provide essential information concerning lipid and pheromone metabolism in Drosophila.
Acknowledgments We thank Professors Jean-Marc Jallon and Leonard Rabinow for helpful discussions and Sabrina Davis for correcting the english. We are grateful to Professor Jaquet and his Laboratory for technical assistance with the functional assay and C.E. Martin for generously providing the DTY001 yeast strain. Funding was provided by the French Ministry of Research and Education (for T.C., L.D., and C.L.) and by the Centre National de la Recherche Scientifique (for C.W.-T.). The nucleotide sequence reported in this paper has been deposited in the GenBank Data under Accession No. AJ178925.
References [1] M. PennanecÕh, L. Bricard, G. Kunesh, J.M. Jallon, Incorporation of fatty acids into cuticular hydrocarbons of male and female Drosophila melanogaster, J. Insect Physiol. 43 (1997) 1111–1116. [2] C. Antony, J.M. Jallon, The chemical basis for sex recognition in Drosophila melanogaster, J. Insect Physiol. 28 (1982) 873–880. [3] C. Antony, T.L. Davis, D.A. Carlson, J.M. Pe´chine´, J.M. Jallon, Compared behavioral responses of male Drosophila melanogaster (Canton S) to natural and synthetic aphrodisiacs, J. Chem. Ecol. 11 (1985) 1617–1629.
[4] C. Wicker-Thomas, C. Henriet, R.R. Dallerac, Partial characterization of a fatty acid desaturase gene in Drosophila melanogaster, Insect Biochem. Mol. Biol. 27 (1997) 963–972. [5] R. Dallerac, C. Labeur, J.M. Jallon, D.C. Knipple, W.L. Roelofs, C. Wicker-Thomas, A D9 desaturase gene with a different substrate specificity is responsible for the cuticular diene hydrocarbon polymorphism in Drosophila melanogaster, Proc. Natl. Acad. Sci. USA 97 (2000) 9449–9454. [6] F.M. Butterworth, Lipids of Drosophila; a new detected lipid in the male, Science 163 (1969) 1256–1257. [7] J.M. Jallon, C. Antony, O. Benamar, Un anti-aphrodisiaque produit par les maˆles de Drosophila melanogaster et transfe´re´ aux femelles lors de la copulation, C.R. Acad. Sci. Paris 292 (1981) 1147–1149. [8] R.J. Bartelt, A. Schaner, L.L. Jackson, Cis-vaccenyl acetate as an aggregation pheromone in Drosophila melanogaster, J. Chem. Ecol. 11 (1985) 1744–1756. [9] D. Pompon, A. Nicolas, Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions, Gene 83 (1989) 15–24. [10] D.A. Toke, C.E. Martin, Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae, J. Biol. Chem. 271 (1996) 18413–18422. [11] C. Labeur, R. Dallerac, C. Wicker-Thomas, Involvement of desat1 gene in the control of Drosophila melanogaster pheromone biosynthesis, Genetica 114 (2002) 269–274. [12] J. Shanklin, E. Whittle, B.G. Fox, Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylane monooxygenase, Biochemistry 33 (1994) 12787–12794. [13] C.S. Oh, D.A. Toke, S. Mandala, C.E. Martin, ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation, J. Biol. Chem. 272 (1997) 17376–17384. [14] P. Tvrdik, R. Westerberg, S. Silve, A. Asadi, A. Jakobsson, B. Cannon, G. Loison, A. Jacobsson, Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids, J. Cell Biol. 149 (2000) 707–718. [15] K. Inagaki, T. Aki, Y. Fukuda, S. Kawamoto, S. Shigeta, K. Ono, O. Suzuki, Identification and expression of a rat fatty acid elongase involved in the biosynthesis of C18 fatty acids, Biosci. Biotechnol. Biochem. 66 (2002) 613–621. [16] Y.A. Moon, N.A. Shah, S. Mohapatra, J.A. Warrington, J.D. Horton, Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins, J. Biol. Chem. 276 (2001) 45358–45366. [17] T. Matsuzaka, H. Shimano, N. Yahagi, T. Yoshikawa, M. Amemiya-Kudo, A.H. Hasty, H. Okazaki, Y. Tamura, Y. Iizuka, K. Ohashi, J. Osuga, A. Takahashi, S. Yato, H. Sone, S. Ishibashi, N. Yamada, Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs, J. Lipid Res. 43 (2002) 911–920.
A new elongase selectively expressed in Drosophila male reproductive system Thomas Chertemps, Line Duportets, Carole Labeur, Claude Wicker-Thomas * Laboratoire de Neurobiologie de lÕApprentissage, de la Me´moire et de la Communication, CNRS UMR 8620, Baˆt. 446, Universite´ Paris-Sud, 91405 ORSAY Ce´dex, France Received 24 May 2005 Available online 13 June 2005
Abstract We have identified an elongase gene, elo68a, which is specifically transcribed in males. We have characterized the elo68a open reading frame, expressed it in fasD elo1D yeast and showed that it could elongate myristoleic and palmitoleic acids, therefore sharing an Elo1 specificity. This elongase was found to be exclusively expressed in male genital system (testis and ejaculatory bulb). Northern blot analysis showed that the elo68a gene was inducible at low temperatures. One P-strain mutant for elo68a and three excision lines for this P-element were subsequently studied. The excision line with only 1% elo68a expression showed decreased levels of vaccenyl acetate, a male pheromone produced in the ejaculatory bulb. The induction of elo68a expression at 21 C was also paralleled with higher vaccenyl acetate production. These results strongly suggest that elo68a is involved in the elongation of short unsaturated fatty acids in males and might play a role in vaccenyl acetate biosynthesis. 2005 Elsevier Inc. All rights reserved. Keywords: Elongase; Drosophila melanogaster; Fatty acid; Elo1; Vaccenyl acetate
In Drosophila, palmitoleic and oleic acids represent half of the total fatty acids [1]. They serve not only as membrane structural components but are also used for energy storage and as bioactive metabolites. Moreover, they are the precursors of sex pheromones, which act during courtship by contact, or at very short distance [2,3]. Pheromone biosynthesis involves a desaturase, Desat1, which introduces a D9 desaturation into palmitic acid [4,5]. Palmitoleic acid is then elongated through elongases, thus leading to vaccenic acid and to long chain x7 monoenic fatty acids which are eventually decarboxylated to give 7-unsaturated hydrocarbons. Vaccenic acid is also the precur*
Corresponding author. Fax: +33 1 69 15 77 26. E-mail address: [email protected] (C. Wicker-Thomas). 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.015
sor of vaccenyl acetate (cVA), a potent nonhydrocarbon male pheromone produced in the ejaculatory bulb and transferred to the female during mating. This compound has been shown both to inhibit male courtship behaviour and to act as an aggregation pheromone [6–8]. In this report, we have characterized an elongase gene (named elo68a) which was specifically expressed in male reproductive system, with a strong expression in testis and a faint expression in ejaculatory bulb. We expressed the elo68a open reading frame in yeast and showed that it encodes an enzyme which can elongate myristoleic and palmitoleic acids. A P-element insertion in the upstream region of elo68a and excision of this transposon were studied. The results suggest that elo68a might be involved in male cVA biosynthesis.
T. Chertemps et al. / Biochemical and Biophysical Research Communications 333 (2005) 1066–1072
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Materials and methods
Results and discussion
Cloning and the expression of Drosophila elo68a in Saccharomyces cerevisiae. 5 0 and 3 0 RACE amplifications were performed to obtain a full-length cDNA sequence, using a 5 0 /3 0 Race kit (Roche Applied Bioscience). The elo68a open reading frame was amplified by RT-PCR from Drosophila adult cDNA using two specific primers (5 0 -CGCGG ATCCCGCATGGCATCACTTGGCATCTCA-3 0 ) and (5 0 -AGCGG TACCTTATATAGGTTTCTTGCCGAC-3 0 ) and cloned directly into a PYEDP vector at BamHI and KpnI sites [9]. The resulting galactoseinducible constructs were introduced into S. cerevisiae strain DTY001 (MATa, fas2::LEU2, elo1::HIS3, leu2-3, leu2-1/2, can1-100, ura-3, ade2-1, his3-11, and his3-15) strain ([10], kindly given by C.E. Martin), using the lithium acetate method. The transformed cells were selected and maintained on uracil-deficient medium: yeast extract/nitrogen/ base (YN) containing 20 lg/ml adenine, 1% tergitol type NP-40, 2% dextrose, and 0.5 mM C14:0. The yeast transformants were cultivated at 30 C, in 50 ml YN containing 20 lg/ml adenine, 1% tergitol, 2% raffinose, and 0.5 mM fatty acid substrate, until the cell density reached 5 · 106 cells/ml. The expression of the transgene was induced by the addition of 2% galactose and additional cultivation for 30 h. Fatty acids were extracted in chloroform/methanol and transformed into methyl esters as previously described. They were then analyzed by GC/MS [5]. Fly stocks and genetics. The control strains, Canton-S and w;TM3 were obtained from the Bloomington Center. The GS11513 line, which harbors a single P-element (GSV6 vector) 502 bp upstream of the elo68a open reading frame, was obtained from the DGSP (Kyoto). To generate excision lines, an imprecise P-element excision strategy was used, as previously described [11]. Drosophila cultures were kept at 25 C in a 12-h light and 12-h dark cycle on standard yeast/cornmeal/agar medium. For experiments, flies were isolated just after emergence and sexes were held separately until use. cVA analysis. cVA of 4-day-old flies was extracted individually in heptane and analyzed by gas liquid chromatography (Perkin Elmer AutoSystem) [5]. Characterization of the excision lines. Three excision lines were characterized at the genomic level. Genomic DNA was amplified by PCR using the primer pair CG32071-5 0 (5 0 -ATGGCATCCAGAATC CGTAGA-3 0 ) and CG32071-3 0 (5 0 -TTAGTAGCGTCTGAAGCGG CG-3 0 ). The locations of these primers are: 867–846 and 435–414 bp upstream of the elo68a open reading frame. Visualisation of the elo68a expression. The elo68a expression in flies was conducted using a vital GFP reporter. The GSV6 vector inserted in the GS11513 line contains basal hsp70 promoter adjacent to a GFP reporter near its 3 0 end. Upon activation of the hsp70 promoter, expression of the labeling GFP gene occurs, which made it possible to visualize the target tissues under the control of 5 0 P adjacent genomic region. The activation of the hsp70 promoter was performed through two series of heat-shock per day (20 min at 37 C, 20 min at 20 C, 20 min at 37 C), from the emergence until 4 days after emergence. Images were visualized and photographed on a Nikon eclipse E800 microscope with a Cool Snap camera. RNA and Northern blot analysis. RNA was extracted from third instar larvae or 4-day-old adults by the Trizol (Gibco) method, according to the procedures recommended by the manufacturer. The total RNA was resolved on a 1% agarose/formaldehyde gel (30 lg/slot) and then transferred to a Positive HCD membrane membrane (Appligene). The blot was prehybridized and hybridized, using the elo68a open reading as a probe [4]. The blots were detected immunochemically with Phototope-Star Chemiluminescent Detection Kit (Biolabs) on Fuji RX film. The film was scanned on a Geldoc 2000 (Bio-Rad) and bands were quantified using the QuantityOne software. Statistics. The data are presented as means ± SEM. An analysis of variance (StudentÕs t test) was used for the statistical analyses, and p < 0.05 was accepted as statistically significant.
Identification of two putative D. melanogaster elongases Two potential elongase genes, designated elo68a (Flybase no. CG32072) and elo68b (Flybase no. CG11801), and another gene with no known homology (Flybase no. CG32071), were identified in the cytological region 68A6 (Fig. 1A). CG32071 and elo68b could not be amplified from either male or female cDNA. elo68a, located 326 nucleotides upstream elo68b, contains three introns and could be amplified by PCR only on male adult cDNA. A mutant line, GS11513, was found to harbor a P-element inserted 502 nucleotides upstream of the initiator codon of CG32072. Structure of the elo68a protein 5 0 and 3 0 RACE amplifications were performed to obtain a full-length cDNA sequence and yielded an open reading frame of 786 nucleotides. The deduced protein, containing 262 amino acids, has 40 more amino acids at the N-terminal end than the protein predicted in Flybase (Fig. 1B). It contains a diiron-oxo binding HXXHH motif in the central part of the protein (residues 140–144). This motif is also present in other fatty acid elongases and is believed to act as Fe-chelating ligand used for the electon transfer for O2-dependent redox reactions [12]. Hydropathy analysis using the Kyte–Doolittle program suggests that it is a transmembrane protein with at least five membrane spanning regions, a characteristic shared by previously identified yeast and mouse elongase enzymes [10,13,14]. Drosophila Elo68a most closely resembles mouse elongase SSC2 (37% identity); the sequence identity of Elo68a to yeast elongases is 29–32% (Fig. 2). Functional expression of elo68a gene in yeast Elongase activities were evaluated in yeast mutant strain DTY001, deficient both in fas2 and elo1 activity. When this mutant was transformed with the empty PYEDP vector and cultivated on medium supplemented with palmitic acid (14:0), significant amounts of 16:0 and 18:0 were present, due to a broad substrate specificity of yeast Elo2 (Fig. 3). Significant levels of 14:1D9, 16:1D9, and 18:1D9 were also generated, due to a D9 desaturation by the yeast endogenous enzyme OLE1. This fatty acid profile did not change after galactose induction of the YEDP vector. To determine whether elo68a encodes a functional enzyme with elongase activity, its open reading frame was cloned into the PYEDP vector (PYEPD-elo68a) and expressed in DTY001yeast (Fig. 3). Without galactose induction, the fatty acid profile did not differ from that obtained with PYEPD alone (with or without induc-
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Fig. 1. The genomic organization of the elo68 locus, the cDNA elo68a sequence and encoded amino acids of the elo68a protein coding sequence. (A) Schematic diagram of the elo68a (CG32072) genomic region. CG11801 and CG32072 encode elongase sequences, but only CG32072 produces detectable transcripts in our analyses. CG32071 is similarly not transcribed. The mutant line (GS11513) contains a P-element inserted 502 nucleotides upstream of the initiator codon of CG32072. (B) The sequences of 961-bp elo68a cDNA and the encoded 262 amino acids are shown. The pair of primer sequences used in the PCR-based cloning is underlined, and the polyadenylation sequence is underlined with a dotted line. The histidine motif is boxed.
tion). After induction of elo68a16:1D11 and 18:1D13 were generated in three subsequent experiments with quantitatively reproducible results, suggesting elongation of endogenous 14:1D9 with 2- and 4-carbon units, respectively. Moreover, a low level of 18:1D11 was observed (1.2%), due to the elongation of 16:1D9. These three unsaturated fatty acids were not detected in yeast transformed with PYEDP alone. If the medium was supplemented with longer chain substrates (18:0, 20:0, 18:1D9 or 18:1D11), no elongation activity could be detected (data not shown). These data suggest that the Elo68a protein has an elongase activity for short chain fatty acids. As far as substrate specificity is concerned, elo68a is more similar to yeast Elo1 as it extends C14 and C16 fatty acids to C16-C18 [10]. Whereas Elo1 is restricted to the elongation of short fatty acids, Elo2 elongates palmitoyl-CoA and stearoyl-CoA up to C22 fatty acids, and Elo3 fatty acids of 20–26 carbons [13]. Animal orthologues acting on fatty acids with chains shorter than C18 have been identified [15–17]. At the protein level,
these mammalian elongases show only 27% identity with Drosophila Elo68a. elo68a expression in wild-type flies Northern blot analysis of elo68a demonstrated specific expression in male adults (Fig. 4A). No detectable signal was obtained in third instar larvae or in female adults; also, no amplification of elo68a could be obtained in female adults and larvae by RT-PCR. This result indicates that elo68a is sex-specifically transcribed. GFP expression under the control of elo68a promoter showed specific labeling in males; this was exclusively found in the reproductive system, with a strong expression in testis, and a faint expression in ejaculatory bulb (Fig. 4B). We then tested the influence of temperature on elo68a expression and found higher expression at 21 C. There was a marked increase in the transcript levels at 21 C (1.5 times more, determined from the Northern blot), compared to 25 and 29 C.
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Fig. 2. Alignment of D. melanogaster Elo68a with yeast (Elo1, Elo2, and Elo3) and mouse (SSC1, SSC2, Cig30, Elovl4, and LCE) elongases. Identical amino acids between two and six sequences are highlighted in light gray, and between seven or nine sequences in dark grey. The conserved histidine motif is boxed. Elo68a is 32%, 31%, 29%, 34%, 37%, 27%, 35%, and 27% identical to Elo1, Elo2, Elo3, SSC1, SSC2, Cig30, Elovl4, and LCE, respectively.
elo68a expression in GS11513 and excised lines The elo68a expression was somewhat increased in GS11513 flies. After excision of the P-element, 60 lines were obtained. Three excision lines were selected for further analysis. The sequences adjacent to the P insertion site in these three excision lines were determined (Fig. 5).
Sequence in ex28 was the same as in wild-type, showing that the excision was precise. In contrast, the P excision in the ex20 and ex25 lines left 44 and 45 nucleotides inserted at the point of the excision, respectively. These insertions contained several nucleotides from the 5 0 and 3 0 end of the P-element and a repeat of eight nucleotides from adjacent genomic sequence.
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Fig. 3. Fatty acid profiles of DTY001 yeast (fasD; elo1D) transformed with an empty PYEDP vector (control) or PYEDPelo68a. Analysis of fatty acid methyl esters by GC-MS with or without induction by the addition of galactose and C14:0 supplementation.
Fig. 4. Expression of elo68a. (A) Northern blot of total RNA from Canton-S strain. RNA was isolated from whole third instar larvae (lane 1) or 4day-old adults reared at 25 C until emergence and then kept at 21 C (lanes 2 and 3), 25 C (lanes 4 and 5) or 29 C (lanes 6 and 7). Thirty micrograms was loaded into each lane of a 1% formaldehyde gel, blotted, and probed with elo68a orf. (B) elo68a expression in male reproductive system. GS11513 males were heatshocked twice a day to induce GFP expression under the control of elo68a promoter and reproductive system dissected at 4 days after emergence. A photograph (400·) of the reproductive system is shown (left); GFP labeling occurs in testis (middle); and ejaculatory bulb (right).
We then investigated elo68a expression in the excision lines and found that elo68a expression was not affected in ex28, but highly reduced in ex25 and ex20, compared to wild-type (Fig. 5 and Table 1).
Vaccenyl acetate and hydrocarbons in GS11513 and excised lines The hydrocarbons of GS11513 and excised flies were affected only in males: a significant increase in 7-trico-
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Fig. 5. Sequences adjacent to the P insertion site in the three excision lines, ex28, ex20 and ex25. The site of insertion at 502 nucleotides (numbered from the first ATG of elo68 open reading frame) is in bold. The sequence found in ex28 is wild-type. The insertion sequences in ex20 and ex25 are underlined. The transcription levels of elo68 were determined from two independent northern experiments and expressed as a percentage of elo68 transcription levels in a w; TM3 wild-type strain.
Table 1 Expression level of elo68 and cVA (ng/male) in flies from control, P or excision lines reared at 25 C (A) and in Canton-S flies maintained at different temperatures from emergence (B) (A) Line
w; TM3
ex20
ex25
ex28
P/TM3
P/P
Expression cVA
100 204 ± 21 a
1 130 ± 32 b
26 259 ± 73 a
80 275 ± 60 a
100 270 ± 41 a
116 226 ± 37 a
(B) Temperature (C)
21
25
29
Expression cVA
150 316 ± 38 b
100 271 ± 15 a
100 286 ± 34 a
Values indicate means ± SEM of 10 independent measurements. In each line, values followed by the same letters are not significantly different at the 0.05 probability level.
sene was observed in GS11513 line and in several excision lines (data not shown). However, this phenotype could not be directly linked to the level of expression of elo68a. As cVA is exclusively produced in male ejaculatory bulb, it was quantified in excision lines and in control flies maintained at different temperatures (Table 1). cVA production was reduced by half in ex20, which shows only 1% elo68a expression. It was not significantly different in the other lines, which were kept at 25 C. However, cVA levels significantly increased in control flies maintained at 21 C, in which elo68a expression was higher. These results suggest that Elo68a might be involved in cVA biosynthesis.
Conclusion This report is the first characterization of an elongase gene in Drosophila. We have found 20 elongase sequences in the Drosophila genome, with homology to yeast and mouse elongases. Several elongases might share similar substrate specificity and the effect on a mutation of one elongase might be partially compensated by the activity of another gene. Nonetheless, we found a mutant phenotype associated with the inactivation of elo68a in male flies: a 50% decrease in levels of vaccenyl acetate occurred when elo68a expression was reduced to 1% of the wild-type level. Moreover, at a relatively lower temperature, elo68a expression is enhanced and an increased production of cVA occurs. As cVA is exclusively
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produced in the ejaculatory bulb, where elo68a is also expressed, it seems likely that elo68a is involved, at least partially, in vaccenyl acetate biosynthesis and could therefore control the elongation step of palmitic acid to vaccenic acid, as the in vitro assay suggests. On the other hand, the reproductive system is not involved in hydrocarbon production. The modification of 7-tricosene in some lines might be an indirect consequence of elo68a activity and/or of other elongases. No significant effect on drosophila total fatty acids was observed either (data not shown), and may be due to the very specific elo68a expression in the male reproductive system. We are continuing to characterize other Drosophila elongases and ultimately, these advances should provide essential information concerning lipid and pheromone metabolism in Drosophila.
Acknowledgments We thank Professors Jean-Marc Jallon and Leonard Rabinow for helpful discussions and Sabrina Davis for correcting the english. We are grateful to Professor Jaquet and his Laboratory for technical assistance with the functional assay and C.E. Martin for generously providing the DTY001 yeast strain. Funding was provided by the French Ministry of Research and Education (for T.C., L.D., and C.L.) and by the Centre National de la Recherche Scientifique (for C.W.-T.). The nucleotide sequence reported in this paper has been deposited in the GenBank Data under Accession No. AJ178925.
References [1] M. PennanecÕh, L. Bricard, G. Kunesh, J.M. Jallon, Incorporation of fatty acids into cuticular hydrocarbons of male and female Drosophila melanogaster, J. Insect Physiol. 43 (1997) 1111–1116. [2] C. Antony, J.M. Jallon, The chemical basis for sex recognition in Drosophila melanogaster, J. Insect Physiol. 28 (1982) 873–880. [3] C. Antony, T.L. Davis, D.A. Carlson, J.M. Pe´chine´, J.M. Jallon, Compared behavioral responses of male Drosophila melanogaster (Canton S) to natural and synthetic aphrodisiacs, J. Chem. Ecol. 11 (1985) 1617–1629.
[4] C. Wicker-Thomas, C. Henriet, R.R. Dallerac, Partial characterization of a fatty acid desaturase gene in Drosophila melanogaster, Insect Biochem. Mol. Biol. 27 (1997) 963–972. [5] R. Dallerac, C. Labeur, J.M. Jallon, D.C. Knipple, W.L. Roelofs, C. Wicker-Thomas, A D9 desaturase gene with a different substrate specificity is responsible for the cuticular diene hydrocarbon polymorphism in Drosophila melanogaster, Proc. Natl. Acad. Sci. USA 97 (2000) 9449–9454. [6] F.M. Butterworth, Lipids of Drosophila; a new detected lipid in the male, Science 163 (1969) 1256–1257. [7] J.M. Jallon, C. Antony, O. Benamar, Un anti-aphrodisiaque produit par les maˆles de Drosophila melanogaster et transfe´re´ aux femelles lors de la copulation, C.R. Acad. Sci. Paris 292 (1981) 1147–1149. [8] R.J. Bartelt, A. Schaner, L.L. Jackson, Cis-vaccenyl acetate as an aggregation pheromone in Drosophila melanogaster, J. Chem. Ecol. 11 (1985) 1744–1756. [9] D. Pompon, A. Nicolas, Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions, Gene 83 (1989) 15–24. [10] D.A. Toke, C.E. Martin, Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae, J. Biol. Chem. 271 (1996) 18413–18422. [11] C. Labeur, R. Dallerac, C. Wicker-Thomas, Involvement of desat1 gene in the control of Drosophila melanogaster pheromone biosynthesis, Genetica 114 (2002) 269–274. [12] J. Shanklin, E. Whittle, B.G. Fox, Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylane monooxygenase, Biochemistry 33 (1994) 12787–12794. [13] C.S. Oh, D.A. Toke, S. Mandala, C.E. Martin, ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation, J. Biol. Chem. 272 (1997) 17376–17384. [14] P. Tvrdik, R. Westerberg, S. Silve, A. Asadi, A. Jakobsson, B. Cannon, G. Loison, A. Jacobsson, Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids, J. Cell Biol. 149 (2000) 707–718. [15] K. Inagaki, T. Aki, Y. Fukuda, S. Kawamoto, S. Shigeta, K. Ono, O. Suzuki, Identification and expression of a rat fatty acid elongase involved in the biosynthesis of C18 fatty acids, Biosci. Biotechnol. Biochem. 66 (2002) 613–621. [16] Y.A. Moon, N.A. Shah, S. Mohapatra, J.A. Warrington, J.D. Horton, Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins, J. Biol. Chem. 276 (2001) 45358–45366. [17] T. Matsuzaka, H. Shimano, N. Yahagi, T. Yoshikawa, M. Amemiya-Kudo, A.H. Hasty, H. Okazaki, Y. Tamura, Y. Iizuka, K. Ohashi, J. Osuga, A. Takahashi, S. Yato, H. Sone, S. Ishibashi, N. Yamada, Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs, J. Lipid Res. 43 (2002) 911–920.