Identification of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells1

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Identi¢cation of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells1 Ju«rgen Seack a , Sanja Perovic a , Vera Gamulin b , Heinz C. Schro«der a , Peter Beutelmann c , Isabel M. Mu«ller a , Werner E.G. Mu«ller a; * a

Institut fu«r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita«t, Duesbergweg 6, D-55099 Mainz, Germany b Institute Rudjer Boskovic, Department of Molecular Genetics, HR-10000 Zagreb, Croatia c Institut fu«r Botanik, Universita«t, Mu«llerweg 6, D-55099 Mainz, Germany Received 22 February 2001; received in revised form 15 May 2001 ; accepted 16 May 2001

Abstract Recently, we reported that cells from the sponge Suberites domuncula respond to ethylene with an increase in intracellular Ca2‡ level [Ca2‡ ]i , and with an upregulation of the expression of (at least) two genes, a Ca2‡ /calmodulin-dependent protein kinase and the potential ethylene-responsive gene, termed SDSNZERR (A. Krasko, H.C. Schro«der, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Mu«ller, W.E.G. Mu«ller, J. Biol. Chem. 274 (1999)). Here, we describe for the first time that also mammalian (3T3) cells respond to ethylene, generated by ethephon, with an immediate and transient, strong increase in [Ca2‡ ]i . Next, the promoter for the sponge SDSNZERR gene was isolated from S. domuncula. It was found that the SDSNZERR gene is positioned adjacent to the SNZ-related gene (SNZ-proximal open reading frame) (SDSNO) and linked, as in Saccharomyces cerevisiae, in a head-to-head manner. Until now, neither homologues nor orthologues of these two genes have been identified in higher metazoan phyla. The full-length genes share a bidirectional promoter. 3T3 cells were transfected with this promoter; the activity of the SDSNZERR promoter was strong and twice as high as that of the SV40 promoter, while the SDSNO promoter was less active. Surprisingly, the activity of the SDSNZERR promoter could not be modulated by ethylene or salicylic acid while it is strongly upregulated, by 4-fold, under serum-starved conditions. It is concluded that the modulation of the level of [Ca2‡ ]i by ethylene in mammalian cells is not correlated with an upregulation of the ethylene-responsive gene SDSNZERR. The data indicate that in mammalian cells, the activity of the SDSNZERR promoter is associated with the repression of serum-mediated growth arrest. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Ethylene ; Ethylene-responsive gene; Calcium metabolism; Gene expression; Promoter ; Signal transduction; Porifera

1. Introduction Metazoa evolved approx. 1000 MYA in the late Proterozoic in the ocean [1]. During that period the atmospheric oxygen concentration and in consequence also the oxygen concentration in the seawater rose from less than 3^10% to 100% of the present situation [2,3]. At the reduced oxygen level the ozone screen was less e¤cient and the ultraviolet (UV) radiation stronger than at the present day. The * Corresponding author. Fax.: +49-6131-392-5243. E-mail address : [email protected] (Werner E. G. Mu«ller). 1 The sequences reported here are deposited in the EMBL data base; the Suberites domuncula gene encoding the SDSNZERR (accession No. AJ277952), the SDSNO gene (AJ277953) as well as the SDSNOc cDNA (AJ277954).

metazoan phylum that was (one of) the ¢rst to diverge from the common ancestor of all Metazoa, the Urmetazoa [4], was the Porifera (sponges). Hence these animals can be considered living fossils [5]. Sponges survived at the presumably higher UV exposure with an e¤cient DNA repair system [6] in an aqueous environment in which alkenes could be produced by photochemical reactions from dissolved organic carbon; ethylene is one major alkene produced [7^9]. At present, the concentration of ethylene in ¢ltered seawater is close to 100 pM [10]. Recently we postulated that sponges can use ethylene as an energy source [11]. Applying the primmorph system from the marine sponge Suberites domuncula (primmorphs are organized aggregates formed from dissociated sponge cells [12]) it could be shown that ethylene at a concentration of 5 WM causes : (i) an upregulation of the intracel-

0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 2 4 6 - 9

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lular Ca2‡ concentration, [Ca2‡ ]i , (ii) a reduction of starvation-induced apoptosis and (iii) an increase in the expression of (at least) two genes, the potential ethyleneresponsive gene, originally termed SDERR, and the gene encoding the Ca2‡ /calmodulin-dependent protein kinase II [11]. The sponge gene, SDERR, is related to the plant stress-induced gene HEVER, which was found to be ethylene-responsive [13], and the SNZ1-3 genes from the yeast Saccharomyces cerevisiae [14]. Therefore, in the present study we termed this sponge gene operationally SDSNZERR. In view of earlier ¢ndings showing that metazoan key proteins are conserved from sponges to Deuterostomia and to a lower extent also to Protostomia (reviewed in [15,16]), in the present study we approached for the ¢rst time the question if elements of the ethylene-dependent signal transduction pathway can be traced also in cells from higher metazoans, here in mammalian cells. The experiments have been performed on two levels. Mouse embryonic 3T3 cells were exposed to ethylene, ¢rst to demonstrate their response with an upregulation of [Ca2‡ ]i , and second to elucidate if the promoter of the S. domuncula SDSNZERR gene is active in 3T3 cells. In the course of this last series of experiments a second gene, SDSNO, was detected and isolated. SNO genes are found in S. cerevisiae adjacent to the SNZ-related genes and named accordingly SNZ-proximal open reading frame (SNO) [17]. Interestingly enough, both in S. domuncula and in S. cerevisiae the two genes, SNZ and SNO, are linked head-tohead and share a bidirectional promoter [17]. Like the SNZ1-3 genes the SNO gene is also highly conserved. In S. cerevisiae the two gene families respond to nutrient limitation [17]. The full-length genes as well as the cDNAs for SDSNZERR and SDSNO have been isolated from S. domuncula and the activity of their promoters has been tested in 3T3 cells after transfection. The activity of the bidirectional promoter of the sponge SDSNZERR^ SDSNO genes was found to be stronger than that of the SV40 promoter. In addition, it could be shown that the activity of the bidirectional promoter is reduced after exposure to salicylic acid and to singlet oxygen, generated by methylene blue in vitro, and is strongly increased after a change of the serum concentration. Ethylene, generated by ethephon, displayed no e¡ect. In previous studies it has been shown that the promoter for the plant HEVER gene is induced by salicylic acid and by ethephon [13]. 2. Materials and methods 2.1. Materials The sources of chemicals and enzymes used were given earlier [18,19]. DMEM medium, methylene blue, salicylic acid and (2-chloroethyl)phosphonic acid (ethephon) were

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obtained from Sigma (Deisenhofen, Germany), Fura 2acetoxymethyl (Fura 2-AM) ester from Molecular Probes (Leiden, The Netherlands) and PCR-DIG (digoxigenin) probe synthesis kit, anti-DIG AP Fab fragments, CDPStar (disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane3,2P-(5P-chloro)tricyclo(3.3.1.13;7 )decan}-4-yl)phenyl phosphate) from Roche Diagnostics (Mannheim, Germany). 2.2. Sponges Specimens of the marine sponge S. domuncula (Porifera, Demospongiae, Hadromerida) were collected in the Northern Adriatic near Rovinj (Croatia) and then kept in aquaria in Mainz (Germany) at a temperature of 17³C. 2.3. Ethephon A water solution of ethephon is stable below a pH of 3.5. Above that value ethephon hydrolyzes under the release of free ethylene and phosphoric acid (H3 PO4 ) [20]. Therefore, ethephon was dissolved in 1Uphosphate-bu¡ered saline (PBS) pH 2.5 in a stock solution of 69 mM and kept at 4³C. The amount of ethylene released into the water was determined in a Shimadzu GC9A apparatus using a FID detector; a column (size 3U600 mm) ¢lled with aluminum oxide was used and the runs were performed at 70³C. 2.4. Loading of 3T3 cells with Fura 2-AM and measurement of intracellular calcium [Ca2‡ ]i was determined by measuring the £uorescence ratio of the Ca2‡ indicator dye Fura 2-AM at 340 and 380 nm as described before [21,22]. `Chambered coverglass' incubation chambers (Lab-Tek, Nunc) were coated with poly-L-lysine. 3T3 cells were loaded in the dark with 8^10 WM Fura 2-AM in DMEM containing 1% fetal calf serum (FCS) and 1% bovine serum albumin at 37³C for 120 min. Subsequently, the cells were washed twice with medium supplemented with 10% FCS and incubated further at 37³C for 60 min. During this period of time cells were loaded with Fura 2-AM (inactive Fura 2), su¤cient for a subsequent hydrolysis to the active Fura 2. For the experiments cells were incubated with ethephon (0.1^1 mM) in Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3 , 2.3 mM CaCl2 , 5.6 mM glucose and 10 mM HEPES; pH 7.4). 3T3 cells were treated with ethephon 5.5 min after starting the determination of [Ca2‡ ]i . The [Ca2‡ ]i level was measured for 13.5 min. As a control 0.5 WM H3 PO4 was used; H3 PO4 might be released during ethylene formation from ethephon [20]. An inverted-stage Olympus IX70 microscope was used for the analysis of the £uorescence as described [22]. Calibration was performed with the `Fura 2 Calcium Imaging Calibration Kit' according to the manufacturer's instructions (Molecular Probes,

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Leiden, The Netherlands). One ratio unit 340/380 nm corresponds to approx. 143 nM [Ca2‡ ]i .

script II SK (Stratagene). DNA sequencing was performed with an automatic DNA sequenator (LI-COR 4200L).

2.5. Construction of the genomic library from S. domuncula

2.7. Isolation of the cDNA and the gene for SNO from the sponge S. domuncula

Specimens of S. domuncula were cut into pieces which were immediately frozen in liquid nitrogen and then ground to a ¢ne powder. The cells were lysed during a 2 h incubation step at 50³C with lysis bu¡er (according to the procedure described in the `Qiagen genomic kit booklet'). Cell debris was removed by centrifugation (5000Ug for 10 min) and the DNA in the supernatant was subsequently puri¢ed by passing through a silica gel matrix (Qiagen genomic tip 500). Genomic DNA was partially digested with Sau3AI (5 min with 1 U) to yield 12^20 kb fragments. Sau3AI overhangs were partially ¢lled in using Klenow enzyme and subsequently ligated into the partially ¢lled in XhoI half-site arms of the cloning vector VFIXII (Stratagene). Ligation reactions were carried out at a vector to insert molar ratio of 2:1. Recombinant phages were encapsulated with MaxPlax packaging extracts (Epicentre Technologies) and used to transform the Escherichia coli strain XL1-Blue MRA (P2), yielding a titer of 1.5U106 pfu/ml. This titer is su¤cient to cover the whole genome of S. domuncula (approx. 1.7U106 kb [23]). After amplifying, the library had a ¢nal titer of 1.7U109 pfu/ml. 2.6. Isolation of the SNO/SNZERR genes Two oligonucleotide primers were designed on the basis of the sponge SDSNZERR cDNA (accession No. Y19159 [11]). The sense primer was 5P-AGCCACCAGTGAGACCCAGAC-3P (nucleotides (nt) 59^79) and the antisense primer was 5P-GATCATAGCAGCTCCCTCAGA3P (nt 506^486). Polymerase chain labeling reaction (PCR) reaction was performed using these primers and the SDSNZERRc cDNA clone [11] in combination with the PCR-DIG probe synthesis kit (Roche Diagnostics). An ampli¢ed 467 bp fragment was obtained and used as a probe. 3U105 pfu of the above-mentioned genomic library of S. domuncula in the VFIXII vector was screened with the isolated probe under stringent conditions using nylon ¢lters. Filters were hybridized overnight at 42³C in DIG Easy-Hyb (Roche Diagnostics) and then washed twice for 5 min with 2USSC, 0.1% NaDodSO4 at room temperature, followed by two additional washes for 15 min with 0.5USSC, 0.1% NaDodSO4 at 68³C. Positive plaques were detected with an alkaline phosphatase-conjugated antiDIG antibody using BCIP/NBT as substrate [24]. Three single positive plaques were isolated, ampli¢ed [25] and the VDNA prepared using the V Midi Kit (Qiagen). Restriction analysis and Southern blotting [25] were performed and two positive SacI clones of 1.0 and 1.8 kb (termed EC1.0 and EC1.8) were subcloned into pBlue-

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The cDNA of the open reading frame SNO was isolated from the cDNA library of S. domuncula [26]. Two oligonucleotide primers were designed on the basis of the sponge genomic SDSNO clone. The forward primer 5PGAGCATTCATTGAACACATACAC-3P (ranging from nt 44 to 66; termed SnoF1 ; see Fig. 4) was used in conjunction with the vector speci¢c primer T7 to amplify the 3P-region of the cDNA. The reverse primer 5P-TCCAGGGATGATCAGACCATC-3P (nt 177^157; SnoR1 ; Fig. 4) was used in conjunction with the vector-speci¢c primer T3 to amplify the 5P-region. The ampli¢cation products were subcloned into pGEM-T; three independent clones were sequenced. The cDNA was termed SDSNOc. To obtain the SNO gene, SDSNO, the SNO cDNA sequence was used to design the primers. A reverse primer 5P-TTGGAATAGAACGCAGTAAAGTA-3P (nt 884^ 906; SnoF3 ; Fig. 4) was used in combination with SnoF1 to amplify the lacking 3P-end of the genomic clone. Since the two genes (SDSNO and SDSNZERR) are overlapping, the complete 5P-end of SDSNO, also including parts of the coding region, have already been isolated with the 5P-end of the SDSNZERR gene (see Fig. 3); the overlap starts at nt 3113 (start of the SDSNO cDNA ; see Figs. 5 and 9). 2.8. Southern blot analysis Ten micrograms of genomic DNA were digested to completion with the indicated restriction nucleases (40 units at 37³C for 8 h followed by a second incubation round with again 40 U of the respective enzyme overnight) and then electrophoresed through a 0.6% (w/v) agarose gel. The DNA was transferred to a positively charged nylon membrane according to the manufacturer's instructions (Roche). Hybridization was performed with a 943 bp long DIG-labeled probe synthesized with the forward primer 5P-AGCCACCAGTGAGACCCAGAC-3P (nt 59^ 79; numbering according to the published sequence [11]) and the reverse primer 5P-TTGTATTAAAGCCTCAGTT3P (nt 981^963) in combination with the SDSNZERRc cDNA. Filters were washed twice at room temperature for 5 min in 2USSC, 1% NaDodSO4 , and then twice at 55³C for 15 min in 0.1USSC, 0.1% NaDodSO4 . Chemiluminescent detection was performed with anti-DIG alkaline phosphatase Fab fragment (Roche) using CDP-Star substrate. 2.9. Sequence comparisons The sequences were analyzed using computer programs

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Fig. 1. E¡ect of ethylene on the [Ca2‡ ]i level in 3T3 cells. Cells were treated with 8 WM (F) or 4 WM of ethylene (b) 5.5 min after starting the experiment; 0.5 WM of H3 PO4 was used as a control (a). The values for the ratios of the 340/380 nm images are shown. Ethylene was generated by addition of ethephon. The arrow marks the time at which ethephon was added to the 3T3 cells. The results are expressed as mean values þ S.E. ; n = 40.

BLAST [27], FASTA [28] and BLOCK SEARCHER [29]. Multiple alignments were performed with CLUSTALW Ver. 1.6 [30]. Phylogenetic trees were constructed on the basis of amino acid (aa) sequence alignments by neighbor joining, as implemented in the `Neighbor' program from the PHYLIP package [31]. The distance matrices were calculated using the Dayho¡ PAM matrix model as described [32]. The degree of support for internal branches was further assessed by bootstrapping [31]. The graphic presentations were prepared with GeneDoc [33]. 2.10. Construction of the luciferase fusion plasmids PCR ampli¢cations with Synergy polymerase (Gencraft) were performed using the plasmid EC1.8 containing the 5P-£anking regions of both genes (SDSNO and SDSNZERR) as template and primers with internal restriction sites. They were used to create and subclone speci¢c putative promoter fragments into the multiple cloning site of the promoterless reporter vector pGL2-basic

(Promega). Two fragments for both orientations (see Fig. 9) of the putative bidirectional promoter were generated using the reverse primers BiPromR1 (5P-GAAGATCTGAGATCTGCCTTGCTTCACAT-3P; nt 34 to 324; Fig. 5) and BiPromR2 (5P-CCCAAGCTTGAAGGAGTTCACAACAGATC-3P; nt 319 to 338; Fig. 4) containing restriction sites for BglII and HindIII (underlined) respectively, in conjunction with the forward primers BiPromF0 (5P-GGGGTACCGTCAGCCAAGGAAAAAGGAGG-3P; nt 3908 to 3888; Fig. 5), BiPromF1 (5P-GGGGTACCGAAGGAGTTCACAACAGATC-3P; nt 3320 to 3301; Fig. 5), BiPromF2 (5PGGGGTACCGAGATCTGCCTTGCTTCACAT-3P; nt 3336 to 3315; Fig. 4) and BiPromF3 (5P-CAGGTACCTTCTCCAATGCCATTAC-3P; nt 3584 to 3560; Fig. 4). The forward primers all contained one KpnI site which is underlined. All constructs were sequenced in both directions in order to check their ¢delity. 2.11. Transfection and luciferase assay The transfection experiments to determine a potential promoter activity were performed with mouse embryonic NIH 3T3 ¢broblasts, which have been obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in DMEM medium supplemented with 10% FCS, 50 units/ml penicillin and 50 Wg/ml streptomycin at 37³C and 5% CO2 in a humidi¢ed incubator. Transient transfections were performed as described earlier [18]. In brief, after reaching approx. 80% con£uence in the cultures, cells were cotransfected with the di¡erent luciferase reporter constructs and the pCMV-L-galactosidase vector (Clontech). Transient transfections were performed with lipofectamine [18,25]. Six hours later the transfection medium was replaced by normal medium. Following an additional incubation for 42 h, the cells were lysed using 250 Wl reporter lysis bu¡er (Promega) and the luciferase activity was measured using the `Luciferase Assay System' (Promega) in a luminometer (LUMAT LB9501; Berthold, Wildbad, Germany). L-Galactosidase activity was determined in a Beckman DU-64 spectrophotometer by the

Fig. 2. Ethylene-induced changes in the distribution of [Ca2‡ ]i in 3T3 cells. Cells were loaded with Fura 2-AM and analyzed with the ratio-imaging system as described in Section 2. The Fura 2 £uorescence was monitored at 340 nm and 380 nm and images were taken 3 min after starting the experiments (a), immediately after addition of ethephon to generate 4 WM of ethylene (time: 5.5 min ; b) and after a total incubation period of 10 min (c). The cells under study are shown by Nomarsky phase contrast interference optics (d). The spectrum color scale ranges from blue (low [Ca2‡ ]i ) to red (high [Ca2‡ ]i ). Scale bar, 50 m.

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Fig. 3. SDSNZERR^SDSNO gene cluster. Schematic representation of the two genes, isolated from S. domuncula : SDSNO and SDSNZERR. The two genes are head-to-head linked and share a bidirectional promoter. The SDSNO gene contains two introns, one is present in the coding region (No. 2) while the second one (No. 1) is present in the 5P-terminal untranslated part. The translated region of the SDSNO gene is separated from the translated region of the SDSNZERR gene by 338 nt. In the SDSNZERR gene ¢ve introns are found. The numbering of the nt of the respective genes starts with +1 at the start codon (see Fig. 4 (SDSNO; here also the nt position of the upstream region of SDSNZERR is indicated (nt 3339)] and Fig. 5 ([SDSNZERR)). The borders of the introns, their sizes and their phases are given. The three SacI sites are indicated : SacI in the SDSNO gene (nt +596^601 with respect to the genomic sequence), as well as the two SacI in the SDSNZERR gene (nt +842^847 and nt +1860^1865). The sequence from the SacI site within the SDSNO gene to the second SacI site within SDSNZERR was obtained by subcloning while the 3P-terminus of SDSNO was completed by PCR.

Fig. 4. Nucleotide sequence and deduced polypeptide sequence of the SDSNO gene. The nucleotide sequence is numbered with +1 beginning with the ATG translation initiation site. The overlapping of the SDSNO gene with the SDSNZERR gene (Fig. 5) is marked in italics. The start ATG for the putative SNZERR_SD polypeptide is indicated (start-SDSNZERR). The deduced aa (in the three letter code) sequence is given below the nt sequence ; the stop codon is indicated (g). The locations of the introns are underlined and the numbers are given in brackets. The start (b) and the terminus (V) of the longest cDNA isolated are marked. In addition, the locations of the primers used for cloning of the complete gene (Sno) as well as for the promoter segments (BiProm) are double underlined ; the SacI site is indicated (in lower case and underlined).

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Fig. 5. Nucleotide and deduced polypeptide sequences of the SDSNZERR gene. The sequence is numbered with +1 beginning with the start ATG site. The stop codon (g), the locations of introns (numbers are given in brackets) as well as the start (b) and the terminus (V (poly(A)-start)) of the longest cDNA isolated are indicated. The three SacI sites are in lower case and underlined. The overlapping of the SDSNZERR gene with the SDSNO gene (Fig. 4) is marked in italics. The start ATG for the putative SNO_SD polypeptide is indicated (start-SDSNO).

coloration assay [34], using o-nitrophenyl-L-D-galactopyranoside as substrate. The luciferase activity was calculated by dividing the RLU (relative light units) value by the OD of the L-Gal colorimetric reaction (in order to normalize the transfection e¤ciency). The activity is given in relation to the expression of the pGL2 promoter vector, which contains the SV40 promoter (positive control). One negative control (transfection with the pGL2-basic vector, containing the luciferase gene but lacking any promoter and enhancer) and one positive control (transfection with

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pGL2 promoter, containing the SV40 promoter) were included. The activity of the GL2-basic vector (basic) was less than 3% with respect to the pGL2 promoter, containing the SV40 promoter. Where indicated ethephon and salicylic acid were added at the indicated concentrations to the cells 40 h after transfection; then the culture was incubated for two more hours. For the generation of singlet oxygen by methylene blue, the dye was added after 40 h and the culture was exposed for 30 min to light of a wavelength between 500

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and 800 nm at room temperature using a HLX 150 W lamp (Osram, Darmstadt, Germany) with an infrared ¢lter WG500 (Schott, Mainz, Germany) as described [35,36]. The experiments were terminated after an additional incubation period of 90 min. In a further series of experiments the cells were transferred for 42 h into medium supplemented with only 1% FCS, or ¢rst for 24 h in medium with 1% FCS and then for 18 h in medium with 10% FCS. 3. Results 3.1. E¡ect of ethylene on [Ca2+]i level In NIH 3T3 ¢broblasts the [Ca2‡ ]i level, expressed as the 340/380 nm ratio, drastically increased from 0.98 þ 0.02 to 1.52 þ 0.13 (corresponding to 77.2 nM of [Ca2‡ ]i ) after addition of 1 mM ethephon; 1 mM ethephon was found to release 8 WM ethylene (Fig. 1). An incubation with 0.5 mM ethephon (4 WM ethylene) resulted in a slightly lower increase; the values of the ratio increased from 1.12 þ 0.01 to 1.48 þ 0.16 (51.5 nM of [Ca2‡ ]i ). The rapid increase in [Ca2‡ ]i is followed by a slower tailing to a ratio of 1.3 (Fig. 1). Lower concentrations of ethylene did not show a signi¢cant upregulation of the [Ca2‡ ]i level (not shown). Controls with 0.5 WM of H3 PO4 (Fig. 1) or without any further addition of a compound showed no signi¢cant changes (not shown). The change of the ratio values can also be documented on the basis of the images; a rapid shift from dark blue to light blue/green/orange/red in all cells was seen after treatment with ethylene (Fig. 2b). Later during the experiment the cells show a light blue image (Fig. 2c). In Fig. 2d the cells are presented by Nomarsky phase contrast interference optics. From these ¢ndings it is concluded that ethylene caused a rapid increase in the [Ca2‡ ]i level in mouse 3T3 cells. Since in a previous study the S. domuncula gene, here named SDSNZERR (previously termed SDERR), displays high similarity to the plant stress-induced gene HEVER, which was found to be ethylene-responsive (see Section 1), the promoter of this gene was isolated and transfected into 3T3 cells. The experiments should clarify if in these cells the SDSNZERR promoter is inducible after exposure to ethylene.

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gene was obtained by PCR as described in Section 2. A schematic representation of the two genes, which are 3127 bp in length which comprises the two genes with the two £anking 3P-untranslated regions (SDSNO: 120 nt ; SDSNZERR: 516 nt), is shown in Fig. 3. The start codons of the two genes, SDSNZERR and SDSNO, are spaced from each other by 338 nt (Fig. 3). They are arranged in a head-to-head organization. The SDSNO gene region from the translation start codon to the stop codon spans 786 bp. The ¢rst intron of the SDSNO gene is localized in the intergenic region (spanning from nt 34 to 378); hence the start ATG is located in the second exon of SDSNO. The second intron, which is located within the transcriptional region is found from nt 347 to 430; splicing of both introns occurs at phase 0 (Figs. 3 and 4). The start (nt 3226) and the end (nt 924) of the longest sequenced cDNA of SDSNO are indicated in Fig. 4. The SDSNZERR gene starts from the intergenic region downstream to reach the poly(A) at nt 1495 (Fig. 5). From comparison with the deduced cDNA, SDSNZERRc ([11] and see below), the locations of introns were determined. All ¢ve introns are present within the codon region and are ^ as seen in the SDSNO gene ^ of small size, ranging from 60 nt to 131 nt (Figs. 3 and 5). With the exception of intron 5, which is of phase 0, all others are phase 1 introns (Fig. 3). To determine the copy number for the S. domuncula SDSNO^SDSNZERR gene cluster Southern analysis of S. domuncula genomic DNA, after digestion with selected restriction enzymes, was probed with the 943 bp long labeled cDNA fragment. Hybridization after digestion with

3.2. Analysis of the SNO/SNZERR genes from S. domuncula As described in Section 2, two oligonucleotide primers designed from selected stretches of the sponge SDSNZERR cDNA were used to obtain a 467 bp fragment which was subsequently used as a probe to isolate the complete SDSNZERR gene and partially the gene of the adjacent SNZ-proximal open reading frame (SNO) termed SDSNO. The complete sequence of the SDSNO

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Fig. 6. Southern blot analysis to determine the copy number for the S. domuncula SDSNO^SDSNZERR gene cluster. S. domuncula genomic DNA (10 Wg) was digested with the restriction enzymes BglII (lane a), KpnI (lane b), NcoI (lane c), PaeI (lane d) and XbaI (lane e). The fragments were subjected to agarose gel electrophoresis and Southern blotting. The blot was probed with the 947 bp long fragment of the DIG-labeled cDNAs, SDSNZERRc. M, size markers (DNA Molecular Weight Marker III from Roche) were run in parallel.

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Fig. 7. SNO and SNO-related sequences. (A) Deduced aa sequence of SNO from S. domuncula (SNOc_SD); it is aligned with SNO1 from S. cerevisiae (SNO1_YEAST, accession No. NP_014065), the hypothetical protein PH1354 from P. horikoshii (HP1354_PH, D71007) and the probable amidotransferase HisH from D. radiodurans (HISH_DERA, G75405). Residues conserved (similar or related with respect to their similar physico-chemical properties) in all sequences are shown in white on black; those in at least two sequences are shaded. The characteristic signature (sign) with the consensus aa (+) are marked. (B) Unrooted phylogenetic cladogram (slanted), constructed after alignment of the (i) bacterial sequences : probable amidotransferase from D. radiodurans (HISH_DERA), hypothetical protein SCL2.12c from Streptomyces coelicolor (SCL_STRCO, AL137778), amidotransferase hisH homologue from Mycobacterium leprae (HISH_MYCLE, S72721), hypothetical protein Rv2604c from Mycobacterium tuberculosis (HP2604_MYCTU, C70570), hypothetical protein YAAE from Bacillus subtilis (YAAE_BACSU, P37528) and hypothetical protein TM0472 from Thermotoga maritima (HP472_THEMA, H72371) ; (ii) archaeal sequences: PH1354 protein from P. horikoshii (HP1354_PH), hypothetical protein HISH from Pyrococcus abyssi (HISH_PYRAB, B75124), hypothetical protein MTH190 from Methanobacterium thermoautotrophicum (HP190_METTHE, F69120), imidazoleglycerol phosphate synthase subunit H homologue from Archaeoglobus fulgidus (HI1648_ARCHAF, E69313), hypothetical protein 281P2 from Sulfolobus solfataricus (281P2_SULSO, Y18930) and hypothetical protein APE0244 from Aeropyrum pernix (HP244_AEROP, C72782); (iii) sequences from yeast: hypothetical protein SPAC222 from Schizosaccharomyces pombe (HP222_SCHPO, AL132798) and the SNO proteins SNO1^SNO3 from S. cerevisiae (SNO1_YEAST to SNO3_YEAST, NP_014065-NP_013813-NP_011126) as well as (iv) from the sponge S. domuncula SNO protein (SNOc_SD). The analysis was performed by neighbor joining as described in Section 2. The numbers at the nodes are an indication of the level of con¢dence ^ given in percentage ^ for the branches as determined by bootstrap analysis (1000 bootstrap replicates).

the restriction enzymes BglII (1.2 and 2.0 kb) and KpnI (1.4 and 3 kb) resulted in two signals (Fig. 6, lanes a and b), while those digested with NcoI (14 kb), PaeI (15 kb) and XbaI (2.0 kb) showed only a single signal (lanes c^e).

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Since in the SDSNZERR gene for both BglII (at nt 1210^ 1215; Fig. 5) and KpnI (at nt 239^244) one digestion site is present, while for the other three enzymes no restriction sites exist, we conclude that at least the SDSNZERR gene

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Fig. 8. The ethylene/stress-inducible proteins, comprising blocks found in the UPF0019 protein family. (A) The sponge polypeptide SNZERR_SD is aligned with the S. cerevisiae SNZ-1 protein (SNZ1_YEAST, accession No. 6323743). The borders of the four blocks, characteristic for the protein family UPF0019, are marked. (B) Unrooted tree (radial) constructed from the sponge and yeast sequences and additionally (i) from fungi: the hypothetical protein from S. pombe (YEM4_SCHPO, O14027), the pyridoxine biosynthesis protein from C. nicotianae (PYRI_CN, AAD13386), the pyridoxine involved protein from Emericella nidulans (PYROA_EN, AAD49809) and the three SNZ members of the stationary phase-induced gene family from S. cerevisiae (SNZ1_YEAST, 6323743; SNZ2_YEAST, 632399, SNZ3_YEAST 6321049); (ii) from Archaea: the hypothetical protein from A. fulgidus (Y508_ARCHAF, O29742), the hypothetical protein from Methanococcus jannaschii (Y677_METJA, Q58090), the ethylene-responsive protein from P. abyssi (ERP_PYRAB, CAB49706), the hypothetical protein from S. solfataricus (HP57730_SULSO, CAB57730); (iii) from bacteria: the singlet oxygen resistance protein from D. radiodurans (SOR_DERA, AAF10938), the conserved hypothetical protein from T. maritima (HP1725_THEMA, AAD35558), the superoxide-inducible protein SOI7 from B. subtilis (YAAD_BACSU, P37527) and the hypothetical protein MTH666 from M. thermoautotrophicum (Y666_METTHE, O26762) ; (iv) from plants: the SOR1 from A. thaliana (SOR1_ARATH, AAC27172), the ethylene-inducible protein from H. brasiliensis (ER1_HEVBR, Q39963) ; as well as (v) from the sponge S. domuncula, the potential ethylene-responsive molecule, ERR (SNZERR_SD, CAB59635 [11]). The analysis has been performed applying the neighbor-joining methods.

of the S. domuncula SDSNO^SDSNZERR gene cluster exists as a single copy gene. 3.3. Analysis of the SNO and SNZERR cDNAs from S. domuncula 3.3.1. SNO cDNA The cDNA SDSNOc, encoding the putative S. domuncula SNO polypeptide SNOc_SD, has a size of 991 nt. The

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predicted open reading frame ranges from nt 151^153 (start methionine) to the stop codon at nt 850^852 encoding a 233 aa long deduced protein sequence (Fig. 7). The calculated Mr of the polypeptide is 25 764 [37]. The sponge SNO polypeptide shows the Prosite signature PS01236 [29] within the segment of the protein aa 49^65 (Fig. 7). The closest similarity of the sponge protein SNOc_SD was found to the corresponding molecules from yeast (example: SNO1_YEAST from S. cerevisiae), Archaea (ex-

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ample: HP1354_PH from Pyrococcus horikoshii) and bacteria (HISH_DERA from Deinococcus radiodurans) with approx. 33% (approx. 50%) identical (identical plus similar) aa in common (Fig. 7A). An unrooted phylogenetic cladogram (slanted) was constructed after alignment of the six bacterial sequences, six archaeal sequences, three sequences from yeast and one from Schizosaccharomyces (Fig. 7B). The tree showed that the sponge protein clusters together with the yeast sequences, a branch which falls together with the one comprising the archaeal sequences; the bacterial sequences are more distantly related to these two branches (Fig. 7B). 3.3.2. SNZERR cDNA A comparative phylogenetic analysis was performed

with the adjacent sponge protein SNZERR_SD. The corresponding cDNA, as well as the description of the deduced Mr 32 704 protein, SNZERR_SD, has been given recently [11]. The sponge sequence comprises four blocks characteristic for the protein family UPF0019 (aa 19^73; aa 81^125; aa 202^250; and aa 251^293) to which the yeast SNZ proteins belong [13,14,29] ; the borders of the four segments are shown in Fig. 8A. The phylogenetic relationship of the sponge SNZERR_SD was established after alignment with the bacterial, archaeal, yeast and plant related sequences. The unrooted tree shows (Fig. 8B) that the sponge protein is separated from all other mentioned taxa and forms a separate branch.

Fig. 9. Schematic representation of the intergenic region between the SDSNZERR and SDSNO genes in S. domuncula and of the promoter activities. (A) Nucleotide sequence of this region, which is delimited by the ATG (SDSNZERR) and TAC (SDSNO) start codons; they are underlined. Within this segment, the starts of the respective cDNAs, which had been isolated, are marked (b); they are compared with the sites which had been identi¢ed by the program `Promoter Prediction by Neural Network' (D). The potential TATA box (TATA), the GC box (GC), the CCAAT (CCA) as well as the ¢ve E boxes (E) are double underlined. (B) Testing of the intergenic region (SDSNZERR^SDSNO) for promotor activity in 3T3 ¢broblasts. Constructs were synthesized by ampli¢cation via PCR as described in Section 2. The following upstream sequences were used to test for promoter activity: two promoter segments for the SDSNZERR gene, ranging from nt 34 (primer BiPromR1 in Fig. 5) to nt 3320 (primer BiPromF1) and from nt 34 to nt 3908 (BiPromF0), respectively. Likewise, two promoter segments for the SDSNO gene were selected: from nt 319 (primer BiPromR2 ; Fig. 4) to nt 3335 (BiPromF2) and from nt 319 to nt 3584 (BiPromF3). The intergenic region (IGR) is 338 nt long. The fragments were digested with KpnI, BglII or HindIII and subcloned into the promoterless vector pGL2-basic. The relative luciferase activities for each construct were measured. The values were correlated to the luciferase activity determined with the plasmid pGL2 promoter, containing the SV40 promoter (values are given in percent). At least six independent experiments, performed in duplicate, have been performed.

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3.4. Potential binding sites for transcription factors in the intergenic region The intergenic region between the start ATG codons of the head-to-head linked SDSNZERR and SDSNO genes has a size of 338 nt (Fig. 9A). For most of the metazoan head-to-head linked genes a bidirectional promoter activity could be detected; like in sponges these intergenic segments range in size from 200 to 400 bp (see Section 4). The starts of the respective cDNAs within the intergenic region, both deduced from the cDNAs and the online service `Promoter Prediction by Neural Network' [38], are shown schematically in Figs. 3 and 9A. The exact transcription initiation site for SDSNZERR was determined experimentally by reverse transcription-PCR and determined to be at nt 350 (not shown; numbers as outlined in Fig. 9A). This ¢nding is interesting, since a `universal' TATA box exists outside the transcription initiation sites; TATAAA at nt 379 to 374 for the SDSNZERR gene and TATATA at nt 376 to 381 for the SDSNO gene. In addition, this region comprises an almost consensus CCAAT (nt 3133 to 3129) and GC box (nt 3118 to 3111) [39]. Furthermore, in the intergenic region ¢ve E boxes (nt 325 to 320; nt 358 to 343; nt 389 to 384; nt 3199 to 3194; and nt3216 to nt3211) are present. Those boxes have been de¢ned as the nuclear consensus sequence CANNTG [40]. In the intergenic region between the SDSNZERR and SDSNO genes no potential ethylene-inducible GCC box (TAAGAGCCCGCC), which is found in plants upstream from ethylene-inducible genes [41,42], is present (Fig. 9A). 3.5. Promoter activity It was not the aim of the present study to elucidate which transcription factors bind to the two sponge promoters. However, e¡orts were undertaken to elucidate their activities in 3T3 cells. In the ¢rst series, the promoter activity of the 338 nt long intergenic region preceding the two genes SDSNZERR as well as of SDSNO was determined in

31

3T3 cells. It was found that the expression of a 320 nt long upstream region of SDSNZERR has a surprisingly high promoter activity, which amounts to 207 þ 19% ; the activity values are correlated to the activity measured with the SV40 promoter containing pGL2 promoter vector which was set to 100% (Fig. 9B). Less active was the 5Pintergenic region of the SDSNO gene with 18 þ 6%. If the segment of the promoter upstream from the SDSNZERR gene was extended over the intergenic region (nt 34 to 3908; Fig. 5) and ranged into the open ready frame (ORF) of the SDSNO gene then the activity was reduced to nearly the half (127 þ 28%; Fig. 9B). If the segment of the promoter upstream from the SDSNO gene was extended into the ORF of the SDSNZERR gene, no signi¢cant di¡erence, with respect to the shorter promoter segment, could be measured (29 þ 3%; Fig. 9B). 3.6. Modulation of the promoter activity In the ¢nal set of experiments, the SDSNZERR promoter activity comprising a 905 nt long upstream region was measured in 3T3 cells. As mentioned, the promoter activity of this segment was 127% after an incubation period of 42 h. Exposure of 3T3 cells to 1 mM ethephon (8 WM ethylene) did not cause a signi¢cant change in the activity (130%; Table 1). Likewise an addition of 1 mM H3 PO4 which might have been released during the ethylene production from ethephon induced no change (120%). Also an incubation of the transfected 3T3 cells with salicylic acid (2 and 20 WM), a compound which induced transcription of the HEVER gene in a plant [13], was without any e¡ect on the sponge promoter in 3T3 cells. A signi¢cant e¡ect was observed with methylene blue. After exposure of this compound at a concentration of 10 WM together with the cells to light, to cause the release of singlet oxygen, the transfected 3T3 cells show a reduction of the promoter activity to 77% (Table 1). At 1 WM methylene blue the compound displayed no signi¢cant e¡ect. In contrast, a strong activation of the sponge SDSNZERR promoter in 3T3 cells was observed if the cells remained under starved conditions (at 1% FCS for 42 h) ; under

Table 1 E¡ect of di¡erent factors on the activity of the 908 nt long promoter of the SDSNZERR gene Compound

Concentration

Promoter activity (%)

None Ethephon Phosphoric acid (pH 7.2) Methylene blue Methylene blue Salicylic acid Salicylic acid Serum starvation Serum starvation (1% FCS; 24 h) then 10% FCS (18 h)

^ 1 mM 1 mM 1 WM 10 WM 2 WM 20 WM 1% FCS 1% FCS+10% FCS

127 þ 28 132 þ 32 120 þ 14 137 þ 20 77 þ 30 135 þ 32 102 þ 33 410 þ 68 233 þ 25

The promoter activity is given in percent to the pGL2 promoter vector, containing the SV40 promoter. The incubation period was 42 h. Means ( þ S.D.) from at least six independent experiments, performed in duplicate, are given.

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those conditions the promoter activity was 410%, compared to the 130% seen in 3T3 cells, cultured in the presence of 10% FCS. If the transfected cells remained for 24 h at 1% FCS and were then transferred to 10% FCS for 18 h, the promoter activity was also signi¢cantly increased (233%). 4. Discussion It is well established that ethylene is a plant hormone, which is induced by endogenous and exogenous (environmental and stress) factors and is involved in plant physiology, e.g. dormancy, growth and development (reviewed in [43]). In plants, ethylene interacts with an ethylene receptor (ETR1) [44] which possesses at the N-terminal domain an ethylene-binding site [45] and exhibits at the Cterminal domain sequence homology to the bacterial twocomponent regulators [46]. In addition, the C-terminal half of the polypeptide contains domains which display homology to histidine kinases and have histidine kinase activity [47]; histidine kinases are found in bacteria and plants. Homologues of ETR1 have been identi¢ed in Arabidopsis thaliana and other plant species [48], implying that they represent a family of conserved receptors in plants. A negative regulator of ethylene responses is the CTR1 gene that encodes a serine/threonine kinase which is closely related to RAF kinases [49]. Like CTR1, EIN3 encodes a nuclear-localized protein [50] which is necessary and su¤cient to activate ethylene responses. In spite of the tremendous advances made in understanding of the ethylene signaling pathway in Arabidopsis, key elements of the transducing mechanism still remain to be elucidated (reviewed in [51^53]). Based on studies with plants it has been concluded that the ethylene signaling system is used in several certain response pathways, e.g. following oxygen radical stress [54,55]. Until very recently [11] the response of metazoan cells to ethylene has not been studied. It was found that cells of sponges, which live in an aqueous milieu that contains this gas (see Section 1), react to ethylene with an increase of the general metabolism, an upregulation of [Ca2‡ ]i and an induction of gene expression [11]. Among the two genes identi¢ed was the plant ethyleneresponsive and stress-induced gene HEVER homologue [13]; induction of HEVER expression is also observed after treatment of the plant Hevea brasiliensis with ethephon and salicylic acid. In the present study we asked if cells from higher metazoan phyla, here the mouse 3T3 cells, also respond to ethylene, released into the medium after addition of ethephon. Ethephon is known to hydrolyze in water resulting in the production of ethylene [20] and to act as an inducer of the ethylene-responsive pathway in plants [56]. Using the same approach as applied previously for sponge cells, the e¡ect of ethylene on the level of [Ca2‡ ]i was measured

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using the Fura 2 imaging method. It could be demonstrated that at ethylene concentrations of 8 WM and 4 WM the mammalian cells strongly react to ethylene. Interesting is the fact that at the same concentration (5 WM) sponge cells react with a shift of the [Ca2‡ ]i . The response of the mammalian cells is even higher (increase of 51^77 nM [Ca2‡ ]i ) than the one seen in sponge cells (14 nM [Ca2‡ ]i ). This result is taken as strong evidence that mammalian cells are sensitive to ethylene. In this study transfection experiments have been performed with the promoter of the proposed sponge ethylene-responsive gene, SDSNZERR, isolated from S. domuncula. The sponge gene displays besides a high similarity to HEVER a considerable sequence similarity to the SNZ genes present in S. cerevisiae [57]; therefore the sponge gene was named SDSNZERR. In turn with this work the sponge SDSNZERR gene was completely isolated and sequenced. To our surprise we found that the SDSNZERR gene is linked head-to-head with a second putative ORF, SDSNO, which shows high sequence similarity to the SNO gene from S. cerevisiae [17]. Such a head-to-head arrangement also exists for the SNO gene with the SNZ gene in S. cerevisiae [17]. This kind of arrangement of the two related genes found in sponges and yeast supports the high conservation of the genes and supports the notion that they may play also important roles in the cell metabolism of these organisms. Until now neither the SNO gene nor the SNZ gene has been found in metazoans other than sponges. The fact that these genes are not present in the Caenorhabditis elegans genome is not too surprising, since the sponge genes display higher similarity to genes from deuterostomians than to protostomians [58]. The screening process for the SDSNZERR gene in mammalian cells is in progress. Since ¢ve out of the seven introns which are found in the SDSNZERR^SDSNO gene cluster belong to the phase 1 introns it can be postulated that the module-coding exons are prone to an exon-shu¥ing process [59]. In Metazoa head-to-head orientation of two (putative) ORFs has been described for example for the actin gene cluster of the tunicate Halocynthia roretzi [60], the human Surf-1/ Surf-2 genes [61], the rat chaperonin 60/chaperonin 10 genes [62] and the human HADHA/HADHB genes [63]. The intergenic region, between the head-to-head oriented SDSNZERR and SDSNO genes, which is 338 nt long was analyzed for potential binding sites for transcription factors. One `universal' TATA box exists which is located outside of both transcripts. In addition, an almost complete CCAAT box as well as a GC box are found. Interesting is the fact that ¢ve E boxes are present in this intergenic region. These boxes are targets of the bHLH (basic helix-loop-helix) transcription factors. The family of these factors include USF-1 and USF-2 (upstream stimulatory factor), members of the myc network, e.g. c-myc, n-myc, l-myc, max and mad (reviewed in [64]). In mammalian systems the bHLH transcription factor

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plays essential roles in regulation of cell proliferation, activation of immunoglobulin expression, di¡erentiation and apoptosis (reviewed in [65]). In S. cerevisiae the SNO and SNZ genes are coregulated and expressed during the transition from the growth to the stationary phase [17]. In the present study it is shown that in S. domuncula the DNA stretch connecting the SDSNZERR^SDSNO genes comprises a bidirectional promoter. In transient expression studies it is shown that the 320 nt long upstream region of the SDSNZERR gene comprises a promoter which has a markedly strong, 2.1fold higher promoter activity in the luciferase assay compared to the SV40 promoter. The promoter of the SDSNO gene is less active and shows only 0.2^0.3-fold the activity of the SV40 promoter. Interesting is the fact that the upstream intergenic segment of SDSNZERR has a signi¢cantly higher promoter activity than the one measured with a longer upstream region including also a part of the coding region of the SDSNO gene. In view of this ¢nding it can be stated that the bidirectional promoter, isolated from the sponge, is recognized by transcription factors present in 3T3 cells; it can be hypothesized that it might also be present in mammalian cells. The characteristic ethylene-inducible GCC box, present in promoters of ethylene-inducible genes [41,42], is missing in the sponge promoters. In a ¢rst study to determine the activity of a sponge promoter in mammalian cells (3T3), the promoter of the protein kinase C gene was analyzed [18]. The results revealed that this promoter is less e¤cient than the SV40 promoter and shows only 50% of its activity. In contrast, the upstream region of the SDSNZERR gene, described here, displays an approx. 2-fold (for the 320 nt long segment) and 1.3-fold (908 nt), respectively, higher activity than the SV40 promoter. Furthermore, and in contrast to the protein kinase C promoter [18], the SDSNZERR promoter can be modulated by extracellular factors. First it is shown that transfected 3T3 cells did not react to exposure to ethylene and to salicylic acid. As mentioned, the SDSNZERR-related gene from plants, HEVER, responds besides to ethylene also to salicylic acid [13]. Furthermore, evidence has been presented that the SOR1 gene which has high sequence similarity to the sponge SDSNZERR gene causes protection of the fungus Cercospora nicotianae against reactive oxygen species [66,67]. Very recently, it could be demonstrated that SOR1 in C. nicotianae is involved in the de novo synthesis of vitamin B-6, in spite of the fact that it shares no sequence similarity to previously identi¢ed pyridoxine synthesis bacterial genes [68]. Until now vitamin B-6 could not be detected in sponge tissue (to be published) and no synthesis of this vitamin has been reported from mammalian cells. The activity of the sponge SDSNZERR promoter can be modulated by singlet oxygen, formed from methylene blue. It is known that singlet oxygen causes a damaging e¡ect on mammalian cells [69]. Hence, we might attribute

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the lower activity of the sponge promoter in response to singlet oxygen to a NF-UB- and/or transcription factor AP-2-dependent downregulation of transcription factors that might be required for the activation of the sponge promoter in mammalian cells (reviewed in [69,70]). The sponge promoter is strongly activated after serum starvation of the cells. A series of mechanisms may be involved in the higher expression of the reporter gene under low serum conditions [71]. One mechanism should be mentioned in particular. In the sponge ¢ve E boxes could be identi¢ed, which are targets for the bHLH transcription factors [64]. Among those is the Stra13 transcriptional repressor [72] which is involved in the modulation of growth arrest [73]. Taken together, mammalian cells, here mouse 3T3 ¢broblasts, respond to ethylene with an increase in [Ca2‡ ]i . In the previous study this increase could be correlated with an upregulation of the ethylene-responsive gene SDSNZERR in S. domuncula [11]. In the approach described here, the activity of the promoter for the sponge SDSNZERR gene was found not to be changed in 3T3 in response to ethylene. Therefore, further studies applying di¡erential display are now in progress to elucidate those gene(s) whose expression is upregulated after exposure to ethylene. However, the promoter activity for the sponge SDSNZERR gene was found to be strongly enhanced if the cells are serum starved. This ¢nding is very remarkable since until now neither homologues nor orthologues of the sponge SDSNZERR gene have been identi¢ed in higher metazoan phyla. Therefore, we conclude that in mammalian cells the expression of a SDSNZERR-related gene is not correlated with ethylene exposure but might be associated with the repression of serum-mediated growth arrest. Acknowledgements This work was supported by grants from the Boehringer Ingelheim Foundation, Fonds der Chemischen Industrie (0161191) and the International Human Frontier Science Program (RG-333/96-M; W.E.G.M.). References [1] [2] [3] [4] [5] [6] [7] [8] [9]

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