- Email: [email protected]
Molecular characterization of Drosophila melanogaster myo-inositol-1-phosphate synthase
Biochimica et Biophysica Acta 1494 (2000) 277^281
www.elsevier.com/locate/bba
Short sequence-paper
Molecular characterization of Drosophila melanogaster myo-inositol-1-phosphate synthase Dongkook Park a , Sangyun Jeong a , Seongsoo Lee a , Sanghee Park b , Jong-Il Kim b , Jeongbin Yim a; * a
National Creative Research Initiative Center for Genetic Reprogramming and School of Biological Sciences, Seoul National University, 56-1 Shinlim-dong, Kwanak-gu, Seoul 151-742, South Korea b Department of Food and Microbial Technology, Seoul Women's University, 126 Kongnung 2-dong, Nowon-gu, Seoul 139-774, South Korea Received 14 January 2000 ; received in revised form 16 March 2000; accepted 22 March 2000
Abstract We have isolated and characterized a cDNA encoding Drosophila melanogaster myo-inositol-1-phosphate synthase (INOS). The deduced Drosophila INOS protein is 50% identical to the Saccharomyces cerevisiae INO1 gene. The putative active site residues are well conserved in Drosophila INOS protein. Southern blot analysis shows that Drosophila INOS gene is a single copy gene. Northern blot analysis reveals that Drosophila INOS gene expresses a 2.0-kb transcript that is more abundant in the head than the body, suggesting that it may be involved in brain function. The recombinant Drosophila INOS protein was expressed in Escherichia coli and the purified protein has proved to have a myo-inositol-1-phosphate synthase activity. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Drosophila; myo-Inositol-1-phosphate synthase; myo-Inositol-1-phosphate
The metabolism of inositol phosphates and inositol phospholipids is a subject of interest due to the role of these molecules in eukaryotic signal transduction. Metabolic products of myo-inositol play critical roles in signal transduction for a wide variety of hormones, growth factors, and neurotransmitters. Most eukaryotic organisms can synthesize myo-inositol-1-phosphate from glucose-6phosphate. The ¢rst step in the myo-inositol biosynthesis is the conversion of glucose-6-phosphate to myo-inositol1-phosphate by myo-inositol-1-phosphate synthase (INOS) [1]. Myo-inositol-1-phosphate is subsequently dephosphorylated by inositol monophosphatase. INOS protein requires NAD , but the net production of NADH is zero. The reaction mechanism involves a three-step process: oxidation with NAD serving as the hydrogen acceptor, condensation to a cyclic product, and reduction with NADH acting as a hydrogen donor [2]. The INO1 locus in Saccharomyces cerevisiae was identi¢ed as the structural gene encoding myo-inositol-1-phosphate synthase [3]. The INO1 gene was isolated by genetic complementation of the INO mutant phenotype and se-
* Corresponding author. Fax: +82-2-871-4315; E-mail : [email protected]
quenced. Following promoter deletion analysis of INO1 mutants, the cis-acting regulatory element, UASINO , was shown to serve as the binding site for the products of the INO2 and INO4 regulatory loci [4^6]. The UASINO element has also been found in the promoter of genes involved in phospholipid biosynthesis, such as the CHO2, OPI3, and PSS1. Phospholipid biosynthesis appears to be highly regulated by myo-inositol through the UASINO element [7,8]. But, many studies of inositol were done about the function of inositol monophosphatase, which is the main target of lithium, the most e¡ective drug for manic depression [9]. In higher eukaryotes, INOS gene is regulated by myo-inositol, salt, and hormones [10^13]. In Drosophila, INOS gene was not reported; but several phospholipid biosynthetic genes are known to have mutant phenotypes of neuronal disruption and developmental abnormalities [14^16]. Some papers have reported that myoinositol is involved in the Wingless signaling pathway [17,18]. Considering the importance of INOS in organisms, the INOS gene is required to de¢ne the role of myo-inositol in phospholipid biosynthesis and development. To isolate the Drosophila INOS gene, we performed low-stringency RT-PCR with degenerate primers to sequence conserved between yeast INO1 and other homo-
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 0 8 5 - 3
BBAEXP 91399 13-11-00
278
D. Park et al. / Biochimica et Biophysica Acta 1494 (2000) 277^281
Fig. 1. The cDNA and deduced amino acid sequence of the Drosophila myo-inositol-1-phosphate synthase (INOS). The cINO3-1 clone was completely sequenced on both strands. The start codon (ATG), stop codon (TGA), and the putative polyadenylation site (TATAAA) are underlined. The GenBank data library accession number of this sequence is AF071103.
BBAEXP 91399 13-11-00
D. Park et al. / Biochimica et Biophysica Acta 1494 (2000) 277^281
279
Fig. 2. The alignment of the putative INOS amino acid sequence with homologs from S. cerevisiae, C. albicans, and A. thaliana. The comparison was carried out using the ClustalW program. The darker boxes show identical regions and the fainter boxes show conserved regions.
logs. The resulting PCR products were homologous to yeast INO1 gene. The cDNA library screening with the PCR product yielded two clones, one of which covers the full-length cDNA (cINOS3-1). The predicted initiation
codon occurs in the context CAGAATG, which is similar to the Kozak consensus translation initiation sequence (CANCATG). The putative Drosophila INOS protein is composed of 565 amino acids (Fig. 1). Its deduced molec-
BBAEXP 91399 13-11-00
280
D. Park et al. / Biochimica et Biophysica Acta 1494 (2000) 277^281
Fig. 3. Northern blot analysis. Two micrograms of each mRNA was electrophoresed on a 1.2% glyceraldehyde agarose gel. mRNA is transferred to a nylon membrane and UV-crosslinked. Hybridization with ExpressHyb solution was performed using the manufacturer's instructions (Clontech). 32 P-labeled random-primed cDNA was used to probe the INOS gene (top) and the rp49 gene as a control (bottom).
ular mass is 62 249 Da and its calculated isoelectric point is 5.82. The Drosophila INOS mRNA is 1996 bp long, including a 5P-untranslated region (UTR) of 138 bp and 3P-UTR of 158 bp, which were con¢rmed by 5P- and 3PRACE. A putative polyadenylation signal is TATAAA at position 1954^1958, which is 115 bp downstream of the ORF. The deduced Drosophila INOS protein is 42, 52 and 47% homologous and 55, 68, and 63% identical to myoinositol-1-phosphate synthase of Saccharomyces cerevisiae, Arabidopsis thaliana, and Candida albicans, respectively (Fig. 2). Compared to the yeast INO1 protein sequence, the Drosophila INOS protein has 38 additional amino acid residues at the C-terminus and the C-terminus of it is more conserved than the N-terminus. Three domains, LWTANSEAF, NGSPQNFVPG, and SYNHLGNNDG, are conserved among species included in Drosophila INOS protein. It suggests that these highly conserved domains may
Fig. 4. Southern blot analysis of Drosophila genomic DNA. The Drosophila genomic DNA (5 Wg) was isolated with DNAzol (Life Technologies) and digested with EcoRI (E), BamHI (B), HindIII (H), and SalI (S). The digested DNAs were electrophoresed on a 0.6% agarose gel, transferred to a nylon membrane, and cross-linked with UV. cDNA (cINOS3-1) was used as a probe for hybridization. The hybridization and washing was performed according to the manufacturer's instructions (Clontech). Size markers are shown on the right.
Fig. 5. The expression of Drosophila INOS protein in E. coli and assay for INOS enzyme activity. (A) SDS-PAGE gel stained with Coomassie blue shows the expression and puri¢cation of recombinant INOS protein. The putative Drosophila INOS was subcloned into the pET vector and expressed in the E. coli BL21(DE3)pLys strain. The crude extract of pET-INOS before (lane 1) and after (lane 2) IPTG induction ; the recombinant INOS protein puri¢ed by Ni -chelating column (lane 3). (B) The puri¢ed Drosophila INOS protein has the enzyme activity. The enzyme activity was measured by the periodate procedure of Barnett et al. [19]. The appropriate enzyme fraction was mixed with 0.3 ml of incubation mixture (5 mM glucose-6-phosphate, 1 mM NAD , 0.2 mM DTT, 2 mM NH4 Cl, and 50 mM Tris^HCl, pH 7.7) and incubated at 37³C for 1 h. The reaction was stopped by the addition of 50 Wl 20% TCA. After centrifugation, 0.1 ml of supernatant was incubated with 0.1 ml of 0.2 M sodium periodate at 37³C for 1 h. The absorbance was measured at 660 nm after adding 2.5 ml of ¢ltered ammonium molybdate^Malachite green reagent (0.42 g (NH4 )6MoO24 4H2 O in a ¢nal volume of 100 ml of 5 N HCl, 0.15 g of Malachite green oxalate in 300 ml of water) and 0.1 ml of 1.5% Tween 20. One unit is de¢ned as 1 Wmol of myo-inositol-1-phosphate formed/h/ml at 37³C.
be the active sites of the INOS proteins and that the INOS proteins evolved from a common ancestor. To examine the extent of Drosophila INOS mRNA expression, we performed a Northern blot analysis of
BBAEXP 91399 13-11-00
D. Park et al. / Biochimica et Biophysica Acta 1494 (2000) 277^281
mRNA obtained from heads and bodies. A transcript of about 2.0 kb was detected and is much more abundant in the head than the body (Fig. 3). It shows that the Drosophila INOS gene plays an important role in the brain. Southern blot analysis of the Drosophila genomic DNA was performed using the isolated cDNA as a probe. A single band in the HindIII- and SalI-digested lanes was observed, suggesting that the Drosophila INOS gene is encoded by a single copy gene (Fig. 4). The deduced Drosophila INOS protein was expressed to see if it had INOS enzyme activity. First, we ampli¢ed the INOS cDNA ORF region with speci¢c primers and with pfu polymerase (Clontech) to minimize misincorporation. The PCR product was subcloned into the pET vector and transformed into Escherichia coli. Expression of the gene product was induced with IPTG and the product that contains the Histidine-tag was puri¢ed with a Ni -chelating column. The molecular weight of the recombinant Drosophila INOS protein estimated from SDS^PAGE was about 60 kDa (Fig. 5A). To test for INOS enzyme activity, we measured the selective release of phosphate residues using the periodate procedure of Barnett et al., which is based on the sensitivity of myo-inositol-1-phosphate and resistance of glucose-6-phosphate to oxidation by periodate [19,20]. The puri¢ed recombinant Drosophila INOS protein contains INOS enzyme activity, as determined by the concentration of released inorganic phosphate (Fig. 5B). The speci¢c activity of the puri¢ed Drosophila INOS protein was 1.86 Wmol/Wg. In this study, we characterized the cDNA encoding INOS gene from D. melanogaster. Our ¢nal goal is to dissect the function of myo-inositol-1-phosphate in Drosophila development. Several inositol mutants involving the easily shocked gene, IPP, and the IP3 receptor have been reported to have phenotypes of altered development and di¡erentiation. Myo-inositol inhibits the Wingless signaling pathway and suppresses the lithium reaction [14^18]. In the future, we will screen for mutants and perform functional studies of myo-inositol. This work is supported by the Creative Research Initiatives of the Korean Ministry of Science and Technology. We thank Changsoo Kim, Sangjoon Kim, Jaeseob Kim for helpful advice on the manuscript and Jung Lee and Jennifer Macke for editorial assistance. Seongsoo Lee is a recipient of the `Brain Korea 21' Fellowship from the Ministry of Education, Republic of Korea. References [1] T. Maeda, F. Eisenberg Jr., Puri¢cation, structure, and catalytic properties of L-myo-inositol-1-phosphate synthase from rat testis, J. Biol. Chem. 255 (1981) 8458^8464.
281
[2] A.L. Majumder, M.D. Johnson, S.A. Henry, 1L-myo-inositol-1-phosphate synthase, Biochim. Biophys. Acta 1348 (1997) 245^256. [3] T.F. Donahue, S.A. Henry, Myo-inositol-1-phosphate synthase; characterization of the enzyme and identi¢cation of its structural gene in yeast, J. Biol. Chem. 256 (1981) 7077^7085. [4] D.M. Nikolo¡, S.A. Henry, Functional characterization of the INO2 gene of Saccharomyces cerevisiae. A positive regulator of phospholipid biosynthesis, J. Biol. Chem. 269 (1994) 7402^7411. [5] M.J. White, J.P. Hirsch, S.A. Henry, The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper, J. Biol. Chem. 266 (1991) 863^872. [6] D.K. Hoshizaki, J.E. Hill, S.A. Henry, The Saccharomyces cerevisiae INO4 gene encodes a small, highly basic protein required for derepression of phospholipid biosynthetic enzymes, J. Biol. Chem. 265 (1990) 4736^4745. [7] F. Paltauf, S.D. Kohlwein, S.A. Henry, Regulation and compartmentalization of lipid synthesis in yeast, in: E.W. Jones, J.R. Pringle, J.R. Broach (Eds.), The Molecular and Cellular Biology of the Yeast Saccharomyces, Vol. 2, Cold Spring Harbor Laboratory Press, Plainview, NY, 1992, pp. 415^500. [8] M.L. Greenberg, J.M. Lopes, Genetic regulation of phospholipid biosynthesis in Saccharomyces cerevisiae, Microbiol. Rev. 60 (1996) 1^20. [9] C.C. Smart, S. Flores, Over-expression of D-myo-inositol-3-phosphate synthase leads to elevated levels of inositol in Arabidopsis, Plant Mol. Biol. 33 (1997) 811^820. [10] R. Hasegawa, F. Eisenberg Jr., Selective hormonal control of myoinositol biosynthesis in reproductive organs and liver of the male rat, Proc. Natl. Acad. Sci. USA 78 (1981) 4863^4866. [11] M.D. Johnson, X. Wang, Di¡erentially expressed forms of 1-L-myoinositol-1-phosphate synthase (EC 5.5.1.4) in Phaseolus vulgaris, J. Biol. Chem. 271 (1996) 17215^17218. [12] D.E. Nelson, G. Rammesmayer, H.J. Bohnert, Regulation of cellspeci¢c inositol metabolism and transport in plant salinity tolerance, Plant Cell 10 (1998) 753^764. [13] P.S. Klein, D.A. Melton, A molecular mechanism for the e¡ect of lithium on development, Proc. Natl. Acad. Sci. USA 93 (1996) 8455^ 8459. [14] J.K. Acharya, P. Labarca, R. Delgado, K. Jalink, C.S. Zuker, Synaptic defects and compensatory regulation of inositol metabolism in inositol polyphosphate 1-phosphatase mutants, Neuron 20 (1998) 1219^1229. [15] P. Pavlidis, M. Ramaswami, M.A. Tanouye, The Drosophila easily shocked gene: a mutation in a phospholipid synthetic pathway causes seizure, neuronal failure, and paralysis, Cell 79 (1994) 23^33. [16] J.K. Acharya, K. Jalink, R.W. Hardy, V. Hartenstein, C.S. Zuker, InsP3 receptor is essential for growth and di¡erentiation but not for vision in Drosophila, Neuron 18 (1997) 881^887. [17] C.M. Hedgepeth, L.J. Conrad, J. Zhang, H. Huang, V.M.Y. Lee, P.S. Klein, Activation of the Wnt signaling pathway: a molecular mechanism for lithium action, Dev. Biol. 185 (1997) 82^91. [18] D.C. Slusarski, V.G. Corces, R.T. Moon, Interaction of Wnt and a Frizzled homologue triggers G-protein-linked phosphatidylinositol signaling, Nature 390 (1997) 410^413. [19] J.E.G. Barnett, R.E. Brice, D.L. Corina, A colorimetric determination of inositol monophosphates as an assay for D-glucose 6-phosphate-1L-myo-inositol-1-phosphate cyclase, Biochem. J. 119 (1970) 183^186. [20] F. Eisenberg Jr., R. Parthasarathy, Measurement of biosynthesis of myo-inositol from glucose 6-phosphate, Methods Enzymol. 141 (1987) 127^143.
BBAEXP 91399 13-11-00
www.elsevier.com/locate/bba
Short sequence-paper
Molecular characterization of Drosophila melanogaster myo-inositol-1-phosphate synthase Dongkook Park a , Sangyun Jeong a , Seongsoo Lee a , Sanghee Park b , Jong-Il Kim b , Jeongbin Yim a; * a
National Creative Research Initiative Center for Genetic Reprogramming and School of Biological Sciences, Seoul National University, 56-1 Shinlim-dong, Kwanak-gu, Seoul 151-742, South Korea b Department of Food and Microbial Technology, Seoul Women's University, 126 Kongnung 2-dong, Nowon-gu, Seoul 139-774, South Korea Received 14 January 2000 ; received in revised form 16 March 2000; accepted 22 March 2000
Abstract We have isolated and characterized a cDNA encoding Drosophila melanogaster myo-inositol-1-phosphate synthase (INOS). The deduced Drosophila INOS protein is 50% identical to the Saccharomyces cerevisiae INO1 gene. The putative active site residues are well conserved in Drosophila INOS protein. Southern blot analysis shows that Drosophila INOS gene is a single copy gene. Northern blot analysis reveals that Drosophila INOS gene expresses a 2.0-kb transcript that is more abundant in the head than the body, suggesting that it may be involved in brain function. The recombinant Drosophila INOS protein was expressed in Escherichia coli and the purified protein has proved to have a myo-inositol-1-phosphate synthase activity. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Drosophila; myo-Inositol-1-phosphate synthase; myo-Inositol-1-phosphate
The metabolism of inositol phosphates and inositol phospholipids is a subject of interest due to the role of these molecules in eukaryotic signal transduction. Metabolic products of myo-inositol play critical roles in signal transduction for a wide variety of hormones, growth factors, and neurotransmitters. Most eukaryotic organisms can synthesize myo-inositol-1-phosphate from glucose-6phosphate. The ¢rst step in the myo-inositol biosynthesis is the conversion of glucose-6-phosphate to myo-inositol1-phosphate by myo-inositol-1-phosphate synthase (INOS) [1]. Myo-inositol-1-phosphate is subsequently dephosphorylated by inositol monophosphatase. INOS protein requires NAD , but the net production of NADH is zero. The reaction mechanism involves a three-step process: oxidation with NAD serving as the hydrogen acceptor, condensation to a cyclic product, and reduction with NADH acting as a hydrogen donor [2]. The INO1 locus in Saccharomyces cerevisiae was identi¢ed as the structural gene encoding myo-inositol-1-phosphate synthase [3]. The INO1 gene was isolated by genetic complementation of the INO mutant phenotype and se-
* Corresponding author. Fax: +82-2-871-4315; E-mail : [email protected]
quenced. Following promoter deletion analysis of INO1 mutants, the cis-acting regulatory element, UASINO , was shown to serve as the binding site for the products of the INO2 and INO4 regulatory loci [4^6]. The UASINO element has also been found in the promoter of genes involved in phospholipid biosynthesis, such as the CHO2, OPI3, and PSS1. Phospholipid biosynthesis appears to be highly regulated by myo-inositol through the UASINO element [7,8]. But, many studies of inositol were done about the function of inositol monophosphatase, which is the main target of lithium, the most e¡ective drug for manic depression [9]. In higher eukaryotes, INOS gene is regulated by myo-inositol, salt, and hormones [10^13]. In Drosophila, INOS gene was not reported; but several phospholipid biosynthetic genes are known to have mutant phenotypes of neuronal disruption and developmental abnormalities [14^16]. Some papers have reported that myoinositol is involved in the Wingless signaling pathway [17,18]. Considering the importance of INOS in organisms, the INOS gene is required to de¢ne the role of myo-inositol in phospholipid biosynthesis and development. To isolate the Drosophila INOS gene, we performed low-stringency RT-PCR with degenerate primers to sequence conserved between yeast INO1 and other homo-
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 0 8 5 - 3
BBAEXP 91399 13-11-00
278
D. Park et al. / Biochimica et Biophysica Acta 1494 (2000) 277^281
Fig. 1. The cDNA and deduced amino acid sequence of the Drosophila myo-inositol-1-phosphate synthase (INOS). The cINO3-1 clone was completely sequenced on both strands. The start codon (ATG), stop codon (TGA), and the putative polyadenylation site (TATAAA) are underlined. The GenBank data library accession number of this sequence is AF071103.
BBAEXP 91399 13-11-00
D. Park et al. / Biochimica et Biophysica Acta 1494 (2000) 277^281
279
Fig. 2. The alignment of the putative INOS amino acid sequence with homologs from S. cerevisiae, C. albicans, and A. thaliana. The comparison was carried out using the ClustalW program. The darker boxes show identical regions and the fainter boxes show conserved regions.
logs. The resulting PCR products were homologous to yeast INO1 gene. The cDNA library screening with the PCR product yielded two clones, one of which covers the full-length cDNA (cINOS3-1). The predicted initiation
codon occurs in the context CAGAATG, which is similar to the Kozak consensus translation initiation sequence (CANCATG). The putative Drosophila INOS protein is composed of 565 amino acids (Fig. 1). Its deduced molec-
BBAEXP 91399 13-11-00
280
D. Park et al. / Biochimica et Biophysica Acta 1494 (2000) 277^281
Fig. 3. Northern blot analysis. Two micrograms of each mRNA was electrophoresed on a 1.2% glyceraldehyde agarose gel. mRNA is transferred to a nylon membrane and UV-crosslinked. Hybridization with ExpressHyb solution was performed using the manufacturer's instructions (Clontech). 32 P-labeled random-primed cDNA was used to probe the INOS gene (top) and the rp49 gene as a control (bottom).
ular mass is 62 249 Da and its calculated isoelectric point is 5.82. The Drosophila INOS mRNA is 1996 bp long, including a 5P-untranslated region (UTR) of 138 bp and 3P-UTR of 158 bp, which were con¢rmed by 5P- and 3PRACE. A putative polyadenylation signal is TATAAA at position 1954^1958, which is 115 bp downstream of the ORF. The deduced Drosophila INOS protein is 42, 52 and 47% homologous and 55, 68, and 63% identical to myoinositol-1-phosphate synthase of Saccharomyces cerevisiae, Arabidopsis thaliana, and Candida albicans, respectively (Fig. 2). Compared to the yeast INO1 protein sequence, the Drosophila INOS protein has 38 additional amino acid residues at the C-terminus and the C-terminus of it is more conserved than the N-terminus. Three domains, LWTANSEAF, NGSPQNFVPG, and SYNHLGNNDG, are conserved among species included in Drosophila INOS protein. It suggests that these highly conserved domains may
Fig. 4. Southern blot analysis of Drosophila genomic DNA. The Drosophila genomic DNA (5 Wg) was isolated with DNAzol (Life Technologies) and digested with EcoRI (E), BamHI (B), HindIII (H), and SalI (S). The digested DNAs were electrophoresed on a 0.6% agarose gel, transferred to a nylon membrane, and cross-linked with UV. cDNA (cINOS3-1) was used as a probe for hybridization. The hybridization and washing was performed according to the manufacturer's instructions (Clontech). Size markers are shown on the right.
Fig. 5. The expression of Drosophila INOS protein in E. coli and assay for INOS enzyme activity. (A) SDS-PAGE gel stained with Coomassie blue shows the expression and puri¢cation of recombinant INOS protein. The putative Drosophila INOS was subcloned into the pET vector and expressed in the E. coli BL21(DE3)pLys strain. The crude extract of pET-INOS before (lane 1) and after (lane 2) IPTG induction ; the recombinant INOS protein puri¢ed by Ni -chelating column (lane 3). (B) The puri¢ed Drosophila INOS protein has the enzyme activity. The enzyme activity was measured by the periodate procedure of Barnett et al. [19]. The appropriate enzyme fraction was mixed with 0.3 ml of incubation mixture (5 mM glucose-6-phosphate, 1 mM NAD , 0.2 mM DTT, 2 mM NH4 Cl, and 50 mM Tris^HCl, pH 7.7) and incubated at 37³C for 1 h. The reaction was stopped by the addition of 50 Wl 20% TCA. After centrifugation, 0.1 ml of supernatant was incubated with 0.1 ml of 0.2 M sodium periodate at 37³C for 1 h. The absorbance was measured at 660 nm after adding 2.5 ml of ¢ltered ammonium molybdate^Malachite green reagent (0.42 g (NH4 )6MoO24 4H2 O in a ¢nal volume of 100 ml of 5 N HCl, 0.15 g of Malachite green oxalate in 300 ml of water) and 0.1 ml of 1.5% Tween 20. One unit is de¢ned as 1 Wmol of myo-inositol-1-phosphate formed/h/ml at 37³C.
be the active sites of the INOS proteins and that the INOS proteins evolved from a common ancestor. To examine the extent of Drosophila INOS mRNA expression, we performed a Northern blot analysis of
BBAEXP 91399 13-11-00
D. Park et al. / Biochimica et Biophysica Acta 1494 (2000) 277^281
mRNA obtained from heads and bodies. A transcript of about 2.0 kb was detected and is much more abundant in the head than the body (Fig. 3). It shows that the Drosophila INOS gene plays an important role in the brain. Southern blot analysis of the Drosophila genomic DNA was performed using the isolated cDNA as a probe. A single band in the HindIII- and SalI-digested lanes was observed, suggesting that the Drosophila INOS gene is encoded by a single copy gene (Fig. 4). The deduced Drosophila INOS protein was expressed to see if it had INOS enzyme activity. First, we ampli¢ed the INOS cDNA ORF region with speci¢c primers and with pfu polymerase (Clontech) to minimize misincorporation. The PCR product was subcloned into the pET vector and transformed into Escherichia coli. Expression of the gene product was induced with IPTG and the product that contains the Histidine-tag was puri¢ed with a Ni -chelating column. The molecular weight of the recombinant Drosophila INOS protein estimated from SDS^PAGE was about 60 kDa (Fig. 5A). To test for INOS enzyme activity, we measured the selective release of phosphate residues using the periodate procedure of Barnett et al., which is based on the sensitivity of myo-inositol-1-phosphate and resistance of glucose-6-phosphate to oxidation by periodate [19,20]. The puri¢ed recombinant Drosophila INOS protein contains INOS enzyme activity, as determined by the concentration of released inorganic phosphate (Fig. 5B). The speci¢c activity of the puri¢ed Drosophila INOS protein was 1.86 Wmol/Wg. In this study, we characterized the cDNA encoding INOS gene from D. melanogaster. Our ¢nal goal is to dissect the function of myo-inositol-1-phosphate in Drosophila development. Several inositol mutants involving the easily shocked gene, IPP, and the IP3 receptor have been reported to have phenotypes of altered development and di¡erentiation. Myo-inositol inhibits the Wingless signaling pathway and suppresses the lithium reaction [14^18]. In the future, we will screen for mutants and perform functional studies of myo-inositol. This work is supported by the Creative Research Initiatives of the Korean Ministry of Science and Technology. We thank Changsoo Kim, Sangjoon Kim, Jaeseob Kim for helpful advice on the manuscript and Jung Lee and Jennifer Macke for editorial assistance. Seongsoo Lee is a recipient of the `Brain Korea 21' Fellowship from the Ministry of Education, Republic of Korea. References [1] T. Maeda, F. Eisenberg Jr., Puri¢cation, structure, and catalytic properties of L-myo-inositol-1-phosphate synthase from rat testis, J. Biol. Chem. 255 (1981) 8458^8464.
281
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