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FebA: a gene for eukaryotic translation initiation factor 4E-binding protein (4E-BP) in Dictyostelium discoideum
Biochimica et Biophysica Acta 1519 (2001) 65^69
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FebA: a gene for eukaryotic translation initiation factor 4E-binding protein (4E-BP) in Dictyostelium discoideum Takahiro Morio a; *, Hiroo Yasukawa b , Hideko Urushihara a , Tamao Saito c , Hiroshi Ochiai c , Ikuo Takeuchi d , Mineko Maeda e , Yoshimasa Tanaka a a Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Molecular Biology Group, Faculty of Engineering, Toyama University, Toyama 930-8555, Japan Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan d Novartis Foundation for the Promotion of Science, Takarazuka, Hyogo 665-0042, Japan e Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan b
c
Received 1 March 2001; received in revised form 1 March 2001 ; accepted 28 March 2001
Abstract We have identified a gene encoding a eukaryotic initiation factor 4E-binding protein (4E-BP) in the EST database of the Dictyostelium cDNA project. The Dictyostelium 4E-BP, designated febA (four e-binding), showed significant similarity to mammalian 4E-BPs. Northern blot analysis revealed that febA was expressed at a high level in the vegetative growth phase but the level of expression decreased during late development. The gene was shown to be non-essential since disruption of the gene had no severe effect; the null mutant proliferated normally and formed normal fruiting bodies. However, strains overexpressing the gene could not be established, suggesting that an excess of FebA protein may have a lethal effect on the cells. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Eukaryotic initiation factor 4E-binding protein; febA; Cap-dependent translation; Dictyostelium
1. Introduction Regulation of translation plays an important role in controlling cell growth and di¡erentiation. In eukaryotes, cap-dependent translation is negatively regulated in part by the eukaryotic initiation factor 4E (eIF-4E)-binding proteins, 4E-BPs, a family of three small acidic proteins. The 4E-BPs bind to eIF4E and block association of the eIF-4E with eIF-4G, which results in inhibition of the formation of the eIF-4F complex and cap-dependent translation [1,2]. Binding of 4E-BPs to eIF4E is reversibly controlled by the phosphorylation states of the 4E-BPs themselves. Hypophosphorylated forms of 4E-BPs strongly interact with eIF-4E, whereas the hyperphosphorylated forms do not [3^5]. Upon stimulation of the cells with serum, growth factors, or hormones, 4E-BP1 is sequentially phosphory-
* Corresponding author. Fax: +81-298-53-6006; E-mail : [email protected]
lated at four highly conserved amino acid residues (Thr37, Thr-46, Ser-65 and Thr-70) and dissociates from eIF4E to relieve the translational inhibition [3,4,6,7]. The cellular slime mold Dictyostelium discoideum is an excellent model organism for studying the molecular mechanisms of cell motility, di¡erentiation and other cellular functions essential for the developmental processes of higher eukaryotes [8]. D. discoideum cells proliferate as unicellular amoeboid cells, but when their food source is exhausted, they aggregate to form a multicellular mass, eventually forming fruiting bodies consisting of two distinct cell types, spores and stalk cells [9]. During development of the fruiting bodies, major changes in the gene expression pro¢le occur in at least four stages [10]. To investigate whether cap-dependent translational regulation is involved in the regulation of gene expression during development, we identi¢ed a cDNA coding for the Dictyostelium homolog of 4E-BP, designated febA (four e-binding). We show here that febA shares a high degree of similarity with mammalian homologs but is not essential for growth and development. We also discuss the possibility that an excess of the FebA protein may exert a lethal e¡ect on the cells.
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 1 9 - 6
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2. Materials and methods 2.1. Identi¢cation and sequencing of febA cDNA By searching for EST data in the Dictyostelium EST database [11], six cDNA clones (SSA681, SSB201, SSL115 SSM607, SSM665 and FC-AK24) were found to encode amino acid sequences with signi¢cant similarity to highly conserved regions of mammalian 4E-BPs. Ambiguous sequences in the ESTs were corrected by comparing them to each other. The sequences of a part of the 5Puntranslated region that did not appear in any of the six cDNA clones was determined by sequencing the entire cDNA insert of clone SSA681. The sequence of the cDNA, designated febA was deposited in the GenBank/EMBL/DDBJ databanks with accession number AB038152. 2.2. Strain, culture methods and development of D. discoideum D. discoideum Ax2 cells were grown in HL5 medium [12] and transformants were grown at 22³C in HL5 medium supplemented with 20 Wg/ml G418 or 10 Wg/ml Blasticidin S. Cells were allowed to develop on an agar plate containing 12 mM Na/K phosphate bu¡er (pH 6.1) at a density of 1.5U106 cells/cm2 . 2.3. Transformation of Dictyostelium cells Cells grown in HL5 medium were harvested at 2U106 cells/ml and resuspended in the electroporation bu¡er (50 mM sucrose and 10 mM sodium phosphate, pH 6.1) at 2U107 cells/ml. The cell suspension (0.8 ml) was mixed with 20 Wg of plasmid DNA, either the plasmid constructed for gene disruption or the one constructed for overexpression of febA and then the cells were subjected to electroporation once at 1.0 kV per 0.4-cm cuvette at 3 WF using a Gene Pulser II with a Capacitance Extender II (Bio-Rad). The cells were transferred to petri-dishes (20U100 mm), grown in 10 ml HL5 for 20 h, and were then grown in HL5 containing 20 Wg/ml G418 or 10 Wg/ml Blasticidin S. 2.4. Vector construction To construct the gene disruption vector, a Blasticidin S resistance (bsr) gene cassette derived from pBsr2 was inserted into the EcoRV site of the SSA681 cDNA plasmid (Fig. 3A) [11,13]. For construction of the overexpression vector, a DNA fragment including the febA protein-coding sequence was ampli¢ed by the polymerase chain reaction (PCR) using the SSA681 plasmid as a template [11]. The primers used were 5P-GGGGGGATCCGAACAACAACAACAATAAC-3P and 5P-GGGGACTAGTGTAATACGACTCACTA-
TAGGGC-3P. The fragment was then digested with BamHI and SpeI and was ligated to the pHK12neo vector plasmid digested with BglII and SpeI (Fig. 4A). pHK12neo is an extrachromosomal vector for Dictyostelium derived from Ddp2 and it has the actin 15 promoter and the 2H3 terminator which are responsible for overexpression (Fig. 4A) [14]. 2.5. Southern and Northern blot analyses For Southern analysis, genomic DNA (1 Wg per lane) was digested with EcoRI, electrophoresed on a 0.8% agarose gel, transferred onto nylon membrane (Hybond-N , Amersham Pharmacia Biotech) and probed with a 32 Plabeled SalI^NotI fragment of the SSA681 cDNA or the coding region of the bsr-cassette used for gene disruption [11,13]. For lower stringent wash, the hybridized membrane was washed with 2USSC (33.3 mM sodium chloride and 33.3 mM sodium citrate) and 1% sodium dodecyl sulfate at room temperature for 5 min, or at 42³C for 30 min. Total RNA was isolated from Dictyostelium cells using the TRIZOL reagent (Gibco BRL Life Technologies), following the manufacturer's instructions. Total RNA (5 Wg per lane) was electrophoresed on a 1% denaturing agarose gel, blotted and hybridized. The procedures followed for preparation of the probes and hybridization were those described previously [15]. The intensity of the signals obtained upon scanning the blots estimated using the Macintosh/Molecular Analyst system. 2.6. PCR ampli¢cation Genomic DNAs extracted from Ax2 and transformant cells were used as the templates for PCR ampli¢cation. The same speci¢c primers as shown in Section 2.4 were used for detection of the febA overexpression construct. These primers were designed to anneal with the ligation site between the vector and the inserted febA sequence so as to avoid ampli¢cation of the chromosomal febA gene [16]. The PCR reactions were performed according to the reagent manufacturer's protocol (Takara Shuzo, Japan). The reaction conditions for each cycle were 94³C for 30 s, 50³C for 30 s and 72³C for 60 s. DNA fragments were ampli¢ed for 30 cycles using a PCR Thermal Cycler MP (Takara Shuzo). 3. Results and discussion 3.1. Analysis of the febA sequence Several cDNA clones which encode a protein homologous to mammalian 4E-BPs were identi¢ed in the Dictyostelium cDNA project. The nucleotide sequence and deduced amino acid sequence are presented in Fig. 1. The cDNA is 714 bp long and consists of a 5P-untranslated
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phosphorylation of other sites, which triggers the release of 4E-BP1 from eIF-4E [7]. 3.2. A knockout strain showed the same phenotype as the wild type
Fig. 1. The nucleotide sequence and predicted amino acid sequence of febA. The EcoRV site used for construction of the gene disruption vector is underlined.
region (UTR) of 357 bp, an open reading frame (ORF) of 312 bp and a 3P-UTR of 45 bp. The long 5P-UTR may have an important role in regulation of translation. We designated the gene as febA (four e-binding). The deduced amino acid sequence of febA was compared with the reported mammalian 4E-BPs and a Drosophila 4E-BP Thor [17]. As shown in Fig. 2, these proteins share signi¢cant similarity in the middle portion of each that contains the eIF4E binding region [18] (Fig. 2, underlined residues). FebA protein contains three out of ¢ve conserved phosphorylation sites (Ser/Thr-Pro) as indicated by arrowheads [19]. It is notable that one of the potential conserved sites missing in FebA corresponds to Thr-37 in human 4E-BP1 and is considered to be a prerequisite for
To examine the e¡ect of loss of the febA on growth and development, a knockout strain was established. The knockout construct was introduced into Ax2 cells to obtain the homologous recombinants. Disruption of the gene was con¢rmed by the increase in size of an EcoRI fragment containing the febA by 1.4 kb corresponding to the size of the bsr gene cassette (Fig. 3B). We further examined the expression of the febA in the wild-type and the disruptant cells by Northern hybridization. In the parental strain Ax2, two mRNA bands were observed in the growth phase and throughout the course of development with a slight decline after 12 h (Fig. 3C). In contrast, no expression of the gene was observed in the null mutant. The null mutant grew, however, and formed the fruiting body normally, indicating that the loss of FebA protein did not have any severe e¡ect on growth and development (data not shown). As in mammals, FebA inhibits translation by binding to eIF-4E and loss of FebA would result in an excess of free eIF-4E potentially recruited to the eIF4F complex. Other factors such as the phosphorylation of eIF-4E or the amount of eIF-4F subunits available would serve to regulate translation normally in the knockout cells. Another possibility is that another member of the 4E-BP family in D. discoideum, not yet identi¢ed, might complement the e¡ect of disruption of febA. To examine the possibility, we have searched on Dictyostelium EST database and genome database (http://www.uni-koeln.de/ dictyostelium/, http://www.sanger.ac.uk/Projects/D_discoideum/, http://dictygenome.bcm.tmc.edu/) and found no other candidate for 4E-BP gene. We also carried out genomic southern hybridization analysis at lower stringency but could ¢nd no additional band, suggesting the absence of another 4E-BP gene (data not shown).
Fig. 2. Alignment of the 4E-BP proteins identi¢ed in D. discoideum (FebA) with that of Drosophila (DmThor) and mammals mouse (m), rat (r) and human (h). Identical and related amino acid residues in the alignment are indicated by ¢lled and open boxes, respectively. The eIF4E binding region is underlined [18]. The conserved phosphorylation sites (Ser/Thr-Pro) in the mammalian proteins are indicated by arrowheads [19]. The accession numbers of the genes are as follows : m4E-BP1, A57396 ; h4E-BP1, S50866; r4E-BP1, A55258 ; m4E-BP2, U75530 ; h4E-BP2, S50867; m4E-BP3, W18851 ; h4EBP3, AF038869; DmThor, AF244353.
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Fig. 3. Establishment of a febA null mutant. (A) Construction of the gene disruption vector. The bsr cassette (¢lled box) was inserted into the EcoRV site of the febA cDNA Fig. 1). The protein coding region and the 5P- and 3P-UTRs of febA are indicated in hatched and open boxes, respectively. (B) Southern hybridization. Genomic DNA (1 Wg) extracted from the Ax2 (lanes 1 and 3) and febA null mutant cells (lanes 2 and 4) was digested with EcoRI, electrophoresed, transferred onto nylon membrane and probed with febA cDNA (lanes 1 and 2) or bsr (lanes 3 and 4). (C) Northern hybridization. Total RNA was extracted from Ax2 and febA null mutant cells at 4-h intervals after starvation. Each lane contained 5 Wg of total RNA.
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T. Morio et al. / Biochimica et Biophysica Acta 1519 (2001) 65^69 Fig. 4. Establishment of cells carrying the febA overexpression vector. (A) Map of the pHK12neo extrachromosomal vector. A DNA fragment including the protein coding region of febA was inserted between the actin 15 promoter (A15P) and the 2H3 terminator (2H3T; see Section 2). (B) PCR ampli¢cation. Primers speci¢c for the overexpression vector were used to examine whether the transformant cells carried the plasmid. The band corresponding to the ampli¢ed DNA fragment is indicated by the arrowhead. Lane 1, 100 bp ladder size markers ; lane 2, genomic DNA extracted from the parental strain was used as the template; lane 3, genomic DNA extracted from a transformant was used as the template. (C) Northern hybridization. Five Wg of total RNA isolated from growing cells was loaded on a denaturing gel, blotted and probed. Lane 1, Ax2 cells; lane 2, the transformant carrying the overexpression vector. The endogenous febA mRNA and the overexpressed febA mRNA are indicated by arrowheads.
6
3.3. Overexpression of febA We examined whether overexpression of febA would a¡ect the growth and development of Dictyostelium cells. We introduced an extrachromosomal plasmid vector which enables overexpression of febA under the control of the actin15 promoter, a well-characterized strong promoter in D. discoideum (Fig. 4A) [16]. By PCR, we examined whether the transformants obtained harbored the construct. A single PCR fragment was obtained only when genomic DNAs extracted from the transformants were used as the template (Fig. 4B). This result con¢rmed that all transformants tested carried the construct. However, we could not obtain transformants overexpressing febA mRNA at high levels. Northern blot analysis showed that most of the transformants expressed febA at a level only 2-fold higher than that of the parental strain as shown in Fig. 4C. Such transformant cells showed the same phenotype as the parental strain (data not shown). We could not establish a strain overexpressing febA at a high level with the extrachromosomal construct employed, in spite of screening a large number of transformants. Similarly, using another construct, an integration vector, we were unable to establish a strain overexpressing febA (data not shown). It is possible that some regulatory mechanism such as post transcriptional regulation of RNA stability might repress higher expression in the transformants. Alternatively, overexpression of FebA may have a lethal e¡ect on the cells and this may be due to the inhibition of cap-dependent translation, as observed in mammalian cells [3]. Acknowledgements We thank Dr. Hidekazu Kuwayama in University of Tsukuba for providing vector plasmid pHK12neo. This study was supported by grants for Research For the Future (JSPS-RFTF96L 00105) from the Japan Society for the Promotion of Science and from the Ministry of Education, Science, Sports and Culture of Japan (08283107).
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References [1] N. Sonenberg, Translational Control, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1996. [2] V.M. Pain, Initiation of protein synthesis in eukaryotic cells, Eur. J. Biochem. 236 (1996) 747^771. [3] A. Pause, G.J. Belsham, A.-C. Gingras, O. Donze, T.A. Lin, J.C. Lawrence Jr., N. Sonenberg, Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5P-cap function, Nature 371 (1994) 762^767. [4] T.A. Lin, T.A. Kong, T. Haystead, A. Pause, G. Belsham, N. Sonenberg, J.C. Lawrence Jr., PHAS-I as a link between mitogen-activated protein kinase and translation initiation, Science 266 (1994) 653^656. [5] A.-C. Gingras, N. Sonenberg, Adenovirus infection inactivates the translational inhibitors 4E-BP1 and 4E-BP2, Virology 237 (1997) 182^186. [6] M. Fleurent, A.-C. Gingras, N. Sonenberg, S. Meloshe, Angiotensin II stimulates phosphorylation of the translational receptor 4E-binding protein 1 by a mitogen-activated protein kinase-dependent mechanism, J. Biol. Chem. 272 (1997) 4006^4012. [7] A.-C. Gingras, S.P. Gygi, B. Raught, R.D. Polakiewicz, R.T. Abraham, M.F. Hoekstra, R. Aebersold, N. Sonenberg, Regulation of 4EBP1 phosphorylation : a novel two-step mechanism, Genes Dev. 13 (1999) 1422^1437. [8] Y. Maeda, K. Inouye, I. Takeuchi (Eds.), Dictyostelium: A Model System for Cell and Developmental Biology, Universal Academy Press, Tokyo, 1997. [9] W.F. Loomis, The Development of Dictyostelium discoideum, Academic Press, New York, 1982. [10] J.A. Cardelli, D.A. Knecht, R. Wunderlich, R.L. Dimond, Major changes in gene expression occur during at least four stages of development of Dictyostelium discoideum, Dev. Biol. 110 (1985) 147^156. [11] T. Morio, H. Urushihara, T. Saito, Y. Ugawa, H. Mizuno, M. Yoshida, R. Yoshino, B.N. Mitra, M. Pi, T. Sato, K. Takemoto, H. Yasukawa, J. Williams, M. Maeda, I. Takeuchi, H. Ochiai, Y. Tanaka, The Dictyostelium developmental cDNA project : generation and analysis of expressed sequence tags from the ¢rst-¢nger stage of development, DNA Res. 5 (1998) 335^340. [12] D.J. Watts, J.M. Ashworth, Growth of myxamoebae of the cellular slime mold Dictyostelium discoideum in axenic culture, Biochem. J. 119 (1970) 171^174. [13] K. Sutoh, A transformation vector for Dictyostelium discoideum with a new selectable marker bsr, Plasmid 30 (1993) 150^154. [14] M.B. Slade, A.C. Chang, K.L. Williams, The sequence and organization of Ddp2, a high-copy-number nuclear plasmid of Dictyostelium discoideum, Plasmid 24 (1990) 195^207. [15] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning : A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [16] D.A. Knecht, S.M. Cohen, W.F. Loomis, H.F. Lodish, Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors, Mol. Cell. Biol. 6 (1986) 3973^3983. [17] A. Bernal, D.A. Kimbrell, Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity, Proc. Natl. Acad. Sci. USA 97 (2000) 6019^6024. [18] S. Mader, H. Lee, A. Pause, N. Sonenberg, The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins, Mol. Cell. Biol. 15 (1995) 4990^4997. [19] P. Fadden, T.A. Haystead, J.C. Lawrence Jr., Identi¢cation of phosphorylation sites in the translational regulator, PHAS-1, that are controlled by insulin and rapamycin in rat adipocytes, J. Biol. Chem. 272 (1997) 10240^10247.
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FebA: a gene for eukaryotic translation initiation factor 4E-binding protein (4E-BP) in Dictyostelium discoideum Takahiro Morio a; *, Hiroo Yasukawa b , Hideko Urushihara a , Tamao Saito c , Hiroshi Ochiai c , Ikuo Takeuchi d , Mineko Maeda e , Yoshimasa Tanaka a a Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Molecular Biology Group, Faculty of Engineering, Toyama University, Toyama 930-8555, Japan Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan d Novartis Foundation for the Promotion of Science, Takarazuka, Hyogo 665-0042, Japan e Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan b
c
Received 1 March 2001; received in revised form 1 March 2001 ; accepted 28 March 2001
Abstract We have identified a gene encoding a eukaryotic initiation factor 4E-binding protein (4E-BP) in the EST database of the Dictyostelium cDNA project. The Dictyostelium 4E-BP, designated febA (four e-binding), showed significant similarity to mammalian 4E-BPs. Northern blot analysis revealed that febA was expressed at a high level in the vegetative growth phase but the level of expression decreased during late development. The gene was shown to be non-essential since disruption of the gene had no severe effect; the null mutant proliferated normally and formed normal fruiting bodies. However, strains overexpressing the gene could not be established, suggesting that an excess of FebA protein may have a lethal effect on the cells. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Eukaryotic initiation factor 4E-binding protein; febA; Cap-dependent translation; Dictyostelium
1. Introduction Regulation of translation plays an important role in controlling cell growth and di¡erentiation. In eukaryotes, cap-dependent translation is negatively regulated in part by the eukaryotic initiation factor 4E (eIF-4E)-binding proteins, 4E-BPs, a family of three small acidic proteins. The 4E-BPs bind to eIF4E and block association of the eIF-4E with eIF-4G, which results in inhibition of the formation of the eIF-4F complex and cap-dependent translation [1,2]. Binding of 4E-BPs to eIF4E is reversibly controlled by the phosphorylation states of the 4E-BPs themselves. Hypophosphorylated forms of 4E-BPs strongly interact with eIF-4E, whereas the hyperphosphorylated forms do not [3^5]. Upon stimulation of the cells with serum, growth factors, or hormones, 4E-BP1 is sequentially phosphory-
* Corresponding author. Fax: +81-298-53-6006; E-mail : [email protected]
lated at four highly conserved amino acid residues (Thr37, Thr-46, Ser-65 and Thr-70) and dissociates from eIF4E to relieve the translational inhibition [3,4,6,7]. The cellular slime mold Dictyostelium discoideum is an excellent model organism for studying the molecular mechanisms of cell motility, di¡erentiation and other cellular functions essential for the developmental processes of higher eukaryotes [8]. D. discoideum cells proliferate as unicellular amoeboid cells, but when their food source is exhausted, they aggregate to form a multicellular mass, eventually forming fruiting bodies consisting of two distinct cell types, spores and stalk cells [9]. During development of the fruiting bodies, major changes in the gene expression pro¢le occur in at least four stages [10]. To investigate whether cap-dependent translational regulation is involved in the regulation of gene expression during development, we identi¢ed a cDNA coding for the Dictyostelium homolog of 4E-BP, designated febA (four e-binding). We show here that febA shares a high degree of similarity with mammalian homologs but is not essential for growth and development. We also discuss the possibility that an excess of the FebA protein may exert a lethal e¡ect on the cells.
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2. Materials and methods 2.1. Identi¢cation and sequencing of febA cDNA By searching for EST data in the Dictyostelium EST database [11], six cDNA clones (SSA681, SSB201, SSL115 SSM607, SSM665 and FC-AK24) were found to encode amino acid sequences with signi¢cant similarity to highly conserved regions of mammalian 4E-BPs. Ambiguous sequences in the ESTs were corrected by comparing them to each other. The sequences of a part of the 5Puntranslated region that did not appear in any of the six cDNA clones was determined by sequencing the entire cDNA insert of clone SSA681. The sequence of the cDNA, designated febA was deposited in the GenBank/EMBL/DDBJ databanks with accession number AB038152. 2.2. Strain, culture methods and development of D. discoideum D. discoideum Ax2 cells were grown in HL5 medium [12] and transformants were grown at 22³C in HL5 medium supplemented with 20 Wg/ml G418 or 10 Wg/ml Blasticidin S. Cells were allowed to develop on an agar plate containing 12 mM Na/K phosphate bu¡er (pH 6.1) at a density of 1.5U106 cells/cm2 . 2.3. Transformation of Dictyostelium cells Cells grown in HL5 medium were harvested at 2U106 cells/ml and resuspended in the electroporation bu¡er (50 mM sucrose and 10 mM sodium phosphate, pH 6.1) at 2U107 cells/ml. The cell suspension (0.8 ml) was mixed with 20 Wg of plasmid DNA, either the plasmid constructed for gene disruption or the one constructed for overexpression of febA and then the cells were subjected to electroporation once at 1.0 kV per 0.4-cm cuvette at 3 WF using a Gene Pulser II with a Capacitance Extender II (Bio-Rad). The cells were transferred to petri-dishes (20U100 mm), grown in 10 ml HL5 for 20 h, and were then grown in HL5 containing 20 Wg/ml G418 or 10 Wg/ml Blasticidin S. 2.4. Vector construction To construct the gene disruption vector, a Blasticidin S resistance (bsr) gene cassette derived from pBsr2 was inserted into the EcoRV site of the SSA681 cDNA plasmid (Fig. 3A) [11,13]. For construction of the overexpression vector, a DNA fragment including the febA protein-coding sequence was ampli¢ed by the polymerase chain reaction (PCR) using the SSA681 plasmid as a template [11]. The primers used were 5P-GGGGGGATCCGAACAACAACAACAATAAC-3P and 5P-GGGGACTAGTGTAATACGACTCACTA-
TAGGGC-3P. The fragment was then digested with BamHI and SpeI and was ligated to the pHK12neo vector plasmid digested with BglII and SpeI (Fig. 4A). pHK12neo is an extrachromosomal vector for Dictyostelium derived from Ddp2 and it has the actin 15 promoter and the 2H3 terminator which are responsible for overexpression (Fig. 4A) [14]. 2.5. Southern and Northern blot analyses For Southern analysis, genomic DNA (1 Wg per lane) was digested with EcoRI, electrophoresed on a 0.8% agarose gel, transferred onto nylon membrane (Hybond-N , Amersham Pharmacia Biotech) and probed with a 32 Plabeled SalI^NotI fragment of the SSA681 cDNA or the coding region of the bsr-cassette used for gene disruption [11,13]. For lower stringent wash, the hybridized membrane was washed with 2USSC (33.3 mM sodium chloride and 33.3 mM sodium citrate) and 1% sodium dodecyl sulfate at room temperature for 5 min, or at 42³C for 30 min. Total RNA was isolated from Dictyostelium cells using the TRIZOL reagent (Gibco BRL Life Technologies), following the manufacturer's instructions. Total RNA (5 Wg per lane) was electrophoresed on a 1% denaturing agarose gel, blotted and hybridized. The procedures followed for preparation of the probes and hybridization were those described previously [15]. The intensity of the signals obtained upon scanning the blots estimated using the Macintosh/Molecular Analyst system. 2.6. PCR ampli¢cation Genomic DNAs extracted from Ax2 and transformant cells were used as the templates for PCR ampli¢cation. The same speci¢c primers as shown in Section 2.4 were used for detection of the febA overexpression construct. These primers were designed to anneal with the ligation site between the vector and the inserted febA sequence so as to avoid ampli¢cation of the chromosomal febA gene [16]. The PCR reactions were performed according to the reagent manufacturer's protocol (Takara Shuzo, Japan). The reaction conditions for each cycle were 94³C for 30 s, 50³C for 30 s and 72³C for 60 s. DNA fragments were ampli¢ed for 30 cycles using a PCR Thermal Cycler MP (Takara Shuzo). 3. Results and discussion 3.1. Analysis of the febA sequence Several cDNA clones which encode a protein homologous to mammalian 4E-BPs were identi¢ed in the Dictyostelium cDNA project. The nucleotide sequence and deduced amino acid sequence are presented in Fig. 1. The cDNA is 714 bp long and consists of a 5P-untranslated
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phosphorylation of other sites, which triggers the release of 4E-BP1 from eIF-4E [7]. 3.2. A knockout strain showed the same phenotype as the wild type
Fig. 1. The nucleotide sequence and predicted amino acid sequence of febA. The EcoRV site used for construction of the gene disruption vector is underlined.
region (UTR) of 357 bp, an open reading frame (ORF) of 312 bp and a 3P-UTR of 45 bp. The long 5P-UTR may have an important role in regulation of translation. We designated the gene as febA (four e-binding). The deduced amino acid sequence of febA was compared with the reported mammalian 4E-BPs and a Drosophila 4E-BP Thor [17]. As shown in Fig. 2, these proteins share signi¢cant similarity in the middle portion of each that contains the eIF4E binding region [18] (Fig. 2, underlined residues). FebA protein contains three out of ¢ve conserved phosphorylation sites (Ser/Thr-Pro) as indicated by arrowheads [19]. It is notable that one of the potential conserved sites missing in FebA corresponds to Thr-37 in human 4E-BP1 and is considered to be a prerequisite for
To examine the e¡ect of loss of the febA on growth and development, a knockout strain was established. The knockout construct was introduced into Ax2 cells to obtain the homologous recombinants. Disruption of the gene was con¢rmed by the increase in size of an EcoRI fragment containing the febA by 1.4 kb corresponding to the size of the bsr gene cassette (Fig. 3B). We further examined the expression of the febA in the wild-type and the disruptant cells by Northern hybridization. In the parental strain Ax2, two mRNA bands were observed in the growth phase and throughout the course of development with a slight decline after 12 h (Fig. 3C). In contrast, no expression of the gene was observed in the null mutant. The null mutant grew, however, and formed the fruiting body normally, indicating that the loss of FebA protein did not have any severe e¡ect on growth and development (data not shown). As in mammals, FebA inhibits translation by binding to eIF-4E and loss of FebA would result in an excess of free eIF-4E potentially recruited to the eIF4F complex. Other factors such as the phosphorylation of eIF-4E or the amount of eIF-4F subunits available would serve to regulate translation normally in the knockout cells. Another possibility is that another member of the 4E-BP family in D. discoideum, not yet identi¢ed, might complement the e¡ect of disruption of febA. To examine the possibility, we have searched on Dictyostelium EST database and genome database (http://www.uni-koeln.de/ dictyostelium/, http://www.sanger.ac.uk/Projects/D_discoideum/, http://dictygenome.bcm.tmc.edu/) and found no other candidate for 4E-BP gene. We also carried out genomic southern hybridization analysis at lower stringency but could ¢nd no additional band, suggesting the absence of another 4E-BP gene (data not shown).
Fig. 2. Alignment of the 4E-BP proteins identi¢ed in D. discoideum (FebA) with that of Drosophila (DmThor) and mammals mouse (m), rat (r) and human (h). Identical and related amino acid residues in the alignment are indicated by ¢lled and open boxes, respectively. The eIF4E binding region is underlined [18]. The conserved phosphorylation sites (Ser/Thr-Pro) in the mammalian proteins are indicated by arrowheads [19]. The accession numbers of the genes are as follows : m4E-BP1, A57396 ; h4E-BP1, S50866; r4E-BP1, A55258 ; m4E-BP2, U75530 ; h4E-BP2, S50867; m4E-BP3, W18851 ; h4EBP3, AF038869; DmThor, AF244353.
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T. Morio et al. / Biochimica et Biophysica Acta 1519 (2001) 65^69
Fig. 3. Establishment of a febA null mutant. (A) Construction of the gene disruption vector. The bsr cassette (¢lled box) was inserted into the EcoRV site of the febA cDNA Fig. 1). The protein coding region and the 5P- and 3P-UTRs of febA are indicated in hatched and open boxes, respectively. (B) Southern hybridization. Genomic DNA (1 Wg) extracted from the Ax2 (lanes 1 and 3) and febA null mutant cells (lanes 2 and 4) was digested with EcoRI, electrophoresed, transferred onto nylon membrane and probed with febA cDNA (lanes 1 and 2) or bsr (lanes 3 and 4). (C) Northern hybridization. Total RNA was extracted from Ax2 and febA null mutant cells at 4-h intervals after starvation. Each lane contained 5 Wg of total RNA.
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T. Morio et al. / Biochimica et Biophysica Acta 1519 (2001) 65^69 Fig. 4. Establishment of cells carrying the febA overexpression vector. (A) Map of the pHK12neo extrachromosomal vector. A DNA fragment including the protein coding region of febA was inserted between the actin 15 promoter (A15P) and the 2H3 terminator (2H3T; see Section 2). (B) PCR ampli¢cation. Primers speci¢c for the overexpression vector were used to examine whether the transformant cells carried the plasmid. The band corresponding to the ampli¢ed DNA fragment is indicated by the arrowhead. Lane 1, 100 bp ladder size markers ; lane 2, genomic DNA extracted from the parental strain was used as the template; lane 3, genomic DNA extracted from a transformant was used as the template. (C) Northern hybridization. Five Wg of total RNA isolated from growing cells was loaded on a denaturing gel, blotted and probed. Lane 1, Ax2 cells; lane 2, the transformant carrying the overexpression vector. The endogenous febA mRNA and the overexpressed febA mRNA are indicated by arrowheads.
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3.3. Overexpression of febA We examined whether overexpression of febA would a¡ect the growth and development of Dictyostelium cells. We introduced an extrachromosomal plasmid vector which enables overexpression of febA under the control of the actin15 promoter, a well-characterized strong promoter in D. discoideum (Fig. 4A) [16]. By PCR, we examined whether the transformants obtained harbored the construct. A single PCR fragment was obtained only when genomic DNAs extracted from the transformants were used as the template (Fig. 4B). This result con¢rmed that all transformants tested carried the construct. However, we could not obtain transformants overexpressing febA mRNA at high levels. Northern blot analysis showed that most of the transformants expressed febA at a level only 2-fold higher than that of the parental strain as shown in Fig. 4C. Such transformant cells showed the same phenotype as the parental strain (data not shown). We could not establish a strain overexpressing febA at a high level with the extrachromosomal construct employed, in spite of screening a large number of transformants. Similarly, using another construct, an integration vector, we were unable to establish a strain overexpressing febA (data not shown). It is possible that some regulatory mechanism such as post transcriptional regulation of RNA stability might repress higher expression in the transformants. Alternatively, overexpression of FebA may have a lethal e¡ect on the cells and this may be due to the inhibition of cap-dependent translation, as observed in mammalian cells [3]. Acknowledgements We thank Dr. Hidekazu Kuwayama in University of Tsukuba for providing vector plasmid pHK12neo. This study was supported by grants for Research For the Future (JSPS-RFTF96L 00105) from the Japan Society for the Promotion of Science and from the Ministry of Education, Science, Sports and Culture of Japan (08283107).
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