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Characterization of the Lupinus luteus cDNA clone encoding ribosomal protein P01
Plant Science 128 (1997) 181 – 190
Characterization of the Lupinus luteus cDNA clone encoding ribosomal protein P01 Katarzyna Mikołajczyk, Maciej Szyman´ski, Jan Barciszewski * Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego 12 /14, 61 -704 Poznan, Poland Received 24 March 1997; received in revised form 12 May 1997; accepted 2 June 1997
Abstract A clone encoding P0 ribosomal protein obtained from a lupin cDNA library has been sequenced and used as a probe in a Northern analysis which revealed a single hybrydization signal of a length sufficient for coding a protein of 322 residues. Southern analysis indicates the presence of several P0 genes in the lupin genome. A putative amino acid sequence encoded by the lupin cDNA clone shows high similarity to eukaryotic proteins P0. Interestingly, the lupin protein contains an RGD motif which can mediate interactions between ribosomes and cell membranes. The phylogenic tree, including amino acid sequences of plant P0 proteins, was generated. © 1997 Elsevier Science Ireland Ltd. Keywords: Ribosome; Ribosomal protein P0; Lupinus luteus
1. Introduction Ribosomes are large nucleoprotein complexes with molecular mass from 2.8 million Da in eubacteria to about 2.5 and 3 million Da in plants and mammals ([1] and references therein). They play a structural and functional role in protein
* Corresponding author. Fax: + 48 61 520532; e-mail: [email protected] 1 Accession numbers: EMBL/Gen Bank X93587, SwissProt Data Libraries P50345.
synthesis. The most important processes carried out by the ribosome are those associated with a formation of the peptide bond. These functions have been mapped to a region named ‘GTPase domain’ which is located close to the large ribosomal subunit central protuberance and forms a specific ‘stalk’ structure [2,3]. In prokaryotic ribosomes, the stalk consists of (L7/12)2L10 protein complex bound cooperatively to protein L11 and 23S rRNA [4,5]. The eukaryotic analogues of the prokaryotic proteins L10, L7 and L12 have been named P0, P1 and P2, respectively [6,7]. In eukaryotic ribosomes P0 protein
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interacts presumably both with the proteins P1 and P2 and with 28S rRNA [8 – 10], but their specific mutual interactions or the participation in elongation processes are not precisely determined. To clarify a mechanism by which the ribosome synthesizes polypeptides in the cell, detailed information is required about a structure of ribosomal proteins, their interactions and function at the molecular level [11]. In order to understand the role of P0 ribosomal protein it is necessary to characterize the structure of this protein and its interactions with 28S rRNA and with other ribosomal proteins. Here we describe the isolation and characteristics of the full length cDNA clone encoding the L. luteus P0 ribosomal protein, Southern and Northern analyses of the lupin genomic DNA and RNA as well as plant P0 ribosomal proteins phylogenetic relationships.
thetic oligodeoxynucleotides complementary to 5% and 3% ends of the L. luteus cDNA open reading frame coding for the ribosomal protein P0 (primer 5%: ATGG-CACCAAAAGCTA; primer 3%: TCACTATT-CATCAAATAGACCG. Northern and Southern analyses were carried out as described [17] and the lupin cDNA coding for protein P0 was used as a probe. It was radiolabelled with [a-32P]dATP using the method described in [18]. Evolutionary analysis was performed using the PHYLIP package. Protein distances matrix was calculated by the PROTDIST programme and the phylogenetic trees were generated using the neighbour-joining method. The consensus tree from the bootstrap analysis of 100 replicates was calculated by the CONSENSE programme [19].
3. Results and discussion
2. Materials and methods A yellow lupin cDNA library prepared from roots was screened with synthetic degenerate oligodeoxynucleotides complementary to the seven C-terminal amino acids MGFGLFD of the eukaryotic P0 and P2 proteins [12] as a probe [13]. Hybridization was carried out in 6× SSC buffer, at 38°C. Three series of the screening were performed, and a cDNA clone containing the L. luteus glutaminyl tRNA synthetase gene was a negative control [14]. Several cDNA clones were obtained and excised as Bluescript SK(− ) vectors. Plasmid DNA was isolated according to the Qiagen Product Guide 1995 manual. Sequencing reactions of both strands were performed with the use of T7 Polymerase sequencing Kit. Lupinus luteus genomic DNA was isolated from 3-day old seedlings as described in [15]. Total RNA was isolated using the method described in [16]. cDNA encoding lupin ribosomal protein P0 (cDNA Ll P0) was amplified by polymerase chain reaction with the use of syn-
The full-length cDNA clone (Fig. 1) is 1393 nt long and has 156 bp 5% and 271 bp 3% flanking regions. In the 5% untranslated region (5% UTR) there is a sequence CTTTCCCC at the immediate 5% end which is characteristic for most eukaryotic ribosomal proteins (rp) mRNAs. This motif may bind a trans-acting factor that accounts for the regulation (and perhaps the coordination) of the translation of ribosomal protein mRNAs [20]. The initiation codon, at position 157, occurs in the context ATCTAUGG which resembles the consensus sequence (A/C)CCAUGG [21]. The open reading frame, starting at the 157 nucleotide encodes a polypeptide of 322 amino acids, with a putative molecular weight of about 34 kDa (Fig. 1). The 3% UTR begins with the UAG stop codon at position 1223 and ends at 1375 nucleotide, by the poly-A stretch. This region contains the hexamer AUCAAA, localized 27 nucleotides 5% to poly-A, which may determine the posttranscriptional cleavage and polyadenylation of the 3% end of the precursor mRNA [7]. The polyadenylation motif differs from the
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Fig. 1.
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Fig. 1. Nucleotide sequence of lupin cDNA encoding ribosomal protein P0. A putative amino acid sequence corresponding to the coding region is written in one letter code.
canonical one, identified as AAUAAA, present in over 50% of plant mRNAs [22]. The lack of a strong consensus sequence may be a result of the presence of other regulatory mechanisms. In plant mRNAs there are cis-acting elements which enhance the polyadenylation signal. These elements are GU-rich [23]. Such motifs are present in the lupin 3% UTR of cDNA clone.
Northern analysis was done in order to confirm the size of expressed mRNA. As a result, one band (Fig. 2) of a length sufficient for coding a protein of 322 residues was detected. This result is consistent with that, obtained for R. nor6egicus ribosomal protein P0 gene organisation [6]. As we know, such analyses have not been done for other eukaryotic P0 proteins.
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Southern analysis of lupin genomic DNA carried out with restriction enzymes: BamHI, HindIII and EcoRI revealed six major bands (Fig. 3, lanes 1, 2) and five bands (Fig. 3, lane 3), respectively. The presence of more than one hybrydizing genomic DNA fragment would indicate that several P0 genes may exist in the lupin genome. Among eukaryotic organisms, P0 genes organization is known only for R. nor6egicus [6]. It revealed several copies of the gene in rat genome. Analyses of other ribosomal protein genes organisation in plants have shown the ex-
Fig. 2. Northern analysis of L. luteus total RNA. A band reflecting hybridization of the radiolabelled cDNA fragment (coding for the lupin ribosomal protein P0) to the lupin RNA, transferred to the nylon Hybond H + membrane as described by Feinberg and Vogelstein (1982) [18]. The positions of molecular length markers in bp are indicated.
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istence of family of genes for each protein, leading to the speculation that plants may have functionally distinct subsets of cytosolic ribosomes [24]. At present, in addition to the lupin P0 protein cDNA, three other cDNA sequences coding for plant ribosomal P0 proteins are known from: C. rubrum [25], O. sati6a [26], and G. max. We aligned them to other eukaryotic proteins P0 sequences (Fig. 4). We also included an EST of G. max protein P0 cDNA sequence (Dr J.A. Rafalski, personal communication). Surprisingly, we found that only plant P0 proteins contain specific motif RGD, which is not present in the other P0 proteins. Up to now this motif has been found in proteins involved in cell adhesion reactions ([27] and references therein). Generally, it is recognized by integrins which are gycoprotein receptors that mediate cell–cell and extracellular matrix–cell adhesion processes. It has been postulated that binding of RGD sequence to integrin might be responsible for high affinity ligand–acceptor interactions [28] and is also recognized by small GTP binding proteins involved in protein kinases regulation [29]. A question arises about the role to be played by RGD sequence in the ribosomal P0 protein. We think this region may be specific for plant ribosomal proteins P0 and could be involved in ribosome-cytoskeleton binding. The amino acid sequence of the yellow lupin ribosomal protein P0, deduced from cDNA, was compared to other P0s available in SwissProt Data Library [30]. This protein appeared to be homologous to higher eukaryotes P0 proteins and shows from 44% identity to the D. discoideum, to and 84% identity to the G. max. The alignment revealed that the lupin protein has conserved regions which are specific for P0 proteins from the other organisms which may be important for the interactions with rRNA and also with other ribosomal proteins [8,31,32]. In the C-terminal region there is a sequence: EEEPAEESDDE. Serine residue localized in this region is phosphorylated [33]. The C-terminal MGFGLFD sequence is highly con-
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Fig. 3. Southern analysis of L. luteus genomic DNA. Bands reflecting hybridization of radiolabelled cDNA (coding for L1P0) to the DNA digested with BamHI (1), HindIII (2), EcoRI(3) and transferred to the nylon Hybond H + membrane as described in Wu et al. (1995) [22]. The positions of molecular length markers in bp are indicated.
served among mammalian and yeast proteins P0 and P2. It is recognized by the antibodies present in the sera of patients with autoimmunological disease systemic lupus erythematosus [12]. This motif is also present in the lupin protein P0, which is not the case of other plant sequences (Fig. 4). The phylogenetic tree generated on the base of ribosomal proteins P0 (L10) sequences (Fig. 5) reflects phylogenetic distances between these proteins from different organisms, showing their molecular evolution. The attempts to find a correct tree using the distance matrix analysis re-
sulted in one diagram, shown in Fig. 5.
Acknowledgements We would like to thank Prof. A.B. Legocki and Dr M. Sikorski of this Institute for kindly providing us L. luteus cDNA library and Dr A. Rafalski of E.I. DuPont, Wilmington, DE., USA, for giving us G. max ESTs. This work was supported within the Committee of Scientific Research (KBN) grant.
Fig. 4. Alignment of aminoacid sequences of ribosomal proteins P0 from eukaryotic organisms; Dictyostelium discoideum, Trypanosoma cruzi, Leischmania chagasi, Saccharomyces cere6isiae (yeast), Chenopodium rubrum, Lupinus luteus (yellow lupin), Glycine max (soybean), Oryza sati6a (rice), Mus musculus (mouse), Rattus nor6egicus (rat), Homo sapiens (human being). The RGD sequence which is specific for plant P0 protein is in bold.
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Fig. 4. (continued)
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Fig. 5. The dendrogram, showing the relationships between the ribosomal proteins P0 sequences derived from protozoa (T. cruzi, L. chagasi ), yeast (S. cere6isae), higher plants (C. rubrum, O. sati6a, G. max, L. Luteus) and mammals (H. sapiens, R. nor6egicus, M. musculus). The numbers indicate percent confirmation of grouping of species to the right of the node by bootstraping analysis with 100 replications.
References [1] A. Verschoor, S. Srivastava, R. Grassucci, J. Frank, Native 3D structure of eukaryotic ribosome, J. Cell Biol. 133 (1996) 495 – 505. [2] A. Lilias, Structural studies of ribosomes, Progr. Biophys. Mol. Biol. 40 (1982) 161–228. [3] L.C. Shimmin, C. Ramirez, A.T. Matheson, P.P. Dennis, Sequence alignment and evolutionary comparison of the L10 equivalent ribosomal protein from archaebacteria, eubacteria and eukaryotes, J. Mol. Evol. 29 (1989) 448– 462. [4] G. Rosendahl, S. Douthwaite, Ribosomal proteins L11 and L10(L12)4 and the antibiotic thiostrepton interact with overlapping regions of the 23S rRNA backbone in the ribosomal GTPase centre, J. Mol. Biol. 234 (1993) 1013 – 1021. [5] G. Rosendahl, S. Douthwaite, Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by their interactions with mutant 23S rRNAs, Nucleic Acids Res. 23 (1995) 2396–2403. [6] I.G. Wool, Y.-L. Chan, A. Glu¨ck, K. Suzuki, The primary structure of rat ribosomal proteins P0, P1 and P2 and a proposal for a uniform nomenclature for mammalian and yeast ribosomal proteins, Biochimie 73 (1991) 861 – 870. [7] I.G. Wool, Y.-L. Chan, A. Glu¨ck, Structure and evolution of mammalian ribosomal proteins, Biochem. Cell Biol. 73 (1995) 933 –947. [8] K. Mitsui, T. Nakagawa, K. Tsurugi, The gene and the primary structure of acidic ribosomal protein A0 from Yeast Saccharomyces cere6isiae which shows partial homology to bacterial ribosomal protein L10, J. Biochem. 106 (1989) 223 – 227. [9] T. Uchiumi, R. Kominami, Direct evidence for interac-
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tion of the conserved GTPase domain within 28 S RNA with mammalian ribosomal acidic phosphoproteins and L12, J. Biol. Chem. 267 (1992) 19179 – 19185. C. Santos, J.P.G. Ballesta, Ribosomal protein P0, contrary to phosphoproteins P1 and P2, is required for ribosome activity and Saccharomyces cere6isiae viability, J. Biol. Chem. 269 (1994) 15689 – 15696. C. Davies, S.T. White, V. Ramakrishnan, The crystal structure of ribosomal protein L14 reveals an important organizational component of the translational apparatus, Structure 4 (1996) 55 – 66. K.B. Elkon, A.P. Parnassa, C.L. Foster, Lupus autoantibodies target ribosomal P proteins, J. Exp. Med. 162 (1985) 459 – 471. Stratagene Instruction Manual, La Jolla, 1993, pp. 7 – 8. M. Siatecka, M. Roz; ek, J. Barciszewski, Isolation and characterization of a cDNA clone containing plant gene of aminoacyl-tRNA synthetase, Plant Physiol. 109 (1995) 1497 – 1499. P.M. Stro´z; ycki, A.B. Legocki, An efficient method of genomic DNA isolation from plant tissues, Acta Biochim. Pol. 42 (1995) 329 – 332. P. Chomczynski, N. Sacchi, Single — step method of RNA isolation by acid guanidinum thiocyanate — phenol — chloroform extraction, Anal. Biochem. 162 (1987) 156 – 159. J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. A.P. Feinberg, B.A. Vogelstein, A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem. 132 (1982) 6 – 13. J. Felsenstein, PHYLIP (Phylogeny Inference Package) version 3.5 c. Distributed by the author. Department of Genetics, University of Washington, Seattle.
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[20] O. Meyuhas, D. Avni, S. Sharma, Translational control, Hershey, Mathews and Sonnenberg, Cold Spring Harbor, NY, 1996, pp. 685 –732. [21] M. Kozak, An analysis of vertebrate mRNA sequences: intimations of translational control, J. Cell. Biol. 115 (1991) 887 – 903. [22] L. Wu, T. Ueda, J. Messing, The formation of mRNA 3%-ends in plants, Plant J. 8 (1995) 323–329. [23] C.P. Joshi, Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis, Nucleic Acids Res. 15 (1987) 9627–9640. [24] V. Sangwan, T.R. Lenvik, J.S. Gantt, The Arabidopsis thaliana ribosomal protein S15 (rig) gene, Biochim. Biophys. Acta 1216 (1993) 221–226. [25] R. Kaldenhoff, G. Richter, Light induction of genes preceding chloroplast differentiation in cultured plant cells, Planta 181 (1990) 220–228. [26] Y. Hihara, M. Umeda, C. Hara, K. Toriyama, H. Uchimiya, Nucleotide sequence of a rice ribosomal phosphoprotein P0 cDNA, Plant Physiol. 105 (1994) 753–754. [27] K.H. Mayo, F. Fan, M.P. Beavers, A. Eckardt, P. Keane, W.J. Hoekstra, P. Andrade-Gordon, RGD induces conformational transition in purified platelet integrin GPIIb/ IIIa — SDS system yelding multiple binding states for
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Characterization of the Lupinus luteus cDNA clone encoding ribosomal protein P01 Katarzyna Mikołajczyk, Maciej Szyman´ski, Jan Barciszewski * Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego 12 /14, 61 -704 Poznan, Poland Received 24 March 1997; received in revised form 12 May 1997; accepted 2 June 1997
Abstract A clone encoding P0 ribosomal protein obtained from a lupin cDNA library has been sequenced and used as a probe in a Northern analysis which revealed a single hybrydization signal of a length sufficient for coding a protein of 322 residues. Southern analysis indicates the presence of several P0 genes in the lupin genome. A putative amino acid sequence encoded by the lupin cDNA clone shows high similarity to eukaryotic proteins P0. Interestingly, the lupin protein contains an RGD motif which can mediate interactions between ribosomes and cell membranes. The phylogenic tree, including amino acid sequences of plant P0 proteins, was generated. © 1997 Elsevier Science Ireland Ltd. Keywords: Ribosome; Ribosomal protein P0; Lupinus luteus
1. Introduction Ribosomes are large nucleoprotein complexes with molecular mass from 2.8 million Da in eubacteria to about 2.5 and 3 million Da in plants and mammals ([1] and references therein). They play a structural and functional role in protein
* Corresponding author. Fax: + 48 61 520532; e-mail: [email protected] 1 Accession numbers: EMBL/Gen Bank X93587, SwissProt Data Libraries P50345.
synthesis. The most important processes carried out by the ribosome are those associated with a formation of the peptide bond. These functions have been mapped to a region named ‘GTPase domain’ which is located close to the large ribosomal subunit central protuberance and forms a specific ‘stalk’ structure [2,3]. In prokaryotic ribosomes, the stalk consists of (L7/12)2L10 protein complex bound cooperatively to protein L11 and 23S rRNA [4,5]. The eukaryotic analogues of the prokaryotic proteins L10, L7 and L12 have been named P0, P1 and P2, respectively [6,7]. In eukaryotic ribosomes P0 protein
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interacts presumably both with the proteins P1 and P2 and with 28S rRNA [8 – 10], but their specific mutual interactions or the participation in elongation processes are not precisely determined. To clarify a mechanism by which the ribosome synthesizes polypeptides in the cell, detailed information is required about a structure of ribosomal proteins, their interactions and function at the molecular level [11]. In order to understand the role of P0 ribosomal protein it is necessary to characterize the structure of this protein and its interactions with 28S rRNA and with other ribosomal proteins. Here we describe the isolation and characteristics of the full length cDNA clone encoding the L. luteus P0 ribosomal protein, Southern and Northern analyses of the lupin genomic DNA and RNA as well as plant P0 ribosomal proteins phylogenetic relationships.
thetic oligodeoxynucleotides complementary to 5% and 3% ends of the L. luteus cDNA open reading frame coding for the ribosomal protein P0 (primer 5%: ATGG-CACCAAAAGCTA; primer 3%: TCACTATT-CATCAAATAGACCG. Northern and Southern analyses were carried out as described [17] and the lupin cDNA coding for protein P0 was used as a probe. It was radiolabelled with [a-32P]dATP using the method described in [18]. Evolutionary analysis was performed using the PHYLIP package. Protein distances matrix was calculated by the PROTDIST programme and the phylogenetic trees were generated using the neighbour-joining method. The consensus tree from the bootstrap analysis of 100 replicates was calculated by the CONSENSE programme [19].
3. Results and discussion
2. Materials and methods A yellow lupin cDNA library prepared from roots was screened with synthetic degenerate oligodeoxynucleotides complementary to the seven C-terminal amino acids MGFGLFD of the eukaryotic P0 and P2 proteins [12] as a probe [13]. Hybridization was carried out in 6× SSC buffer, at 38°C. Three series of the screening were performed, and a cDNA clone containing the L. luteus glutaminyl tRNA synthetase gene was a negative control [14]. Several cDNA clones were obtained and excised as Bluescript SK(− ) vectors. Plasmid DNA was isolated according to the Qiagen Product Guide 1995 manual. Sequencing reactions of both strands were performed with the use of T7 Polymerase sequencing Kit. Lupinus luteus genomic DNA was isolated from 3-day old seedlings as described in [15]. Total RNA was isolated using the method described in [16]. cDNA encoding lupin ribosomal protein P0 (cDNA Ll P0) was amplified by polymerase chain reaction with the use of syn-
The full-length cDNA clone (Fig. 1) is 1393 nt long and has 156 bp 5% and 271 bp 3% flanking regions. In the 5% untranslated region (5% UTR) there is a sequence CTTTCCCC at the immediate 5% end which is characteristic for most eukaryotic ribosomal proteins (rp) mRNAs. This motif may bind a trans-acting factor that accounts for the regulation (and perhaps the coordination) of the translation of ribosomal protein mRNAs [20]. The initiation codon, at position 157, occurs in the context ATCTAUGG which resembles the consensus sequence (A/C)CCAUGG [21]. The open reading frame, starting at the 157 nucleotide encodes a polypeptide of 322 amino acids, with a putative molecular weight of about 34 kDa (Fig. 1). The 3% UTR begins with the UAG stop codon at position 1223 and ends at 1375 nucleotide, by the poly-A stretch. This region contains the hexamer AUCAAA, localized 27 nucleotides 5% to poly-A, which may determine the posttranscriptional cleavage and polyadenylation of the 3% end of the precursor mRNA [7]. The polyadenylation motif differs from the
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Fig. 1.
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Fig. 1. Nucleotide sequence of lupin cDNA encoding ribosomal protein P0. A putative amino acid sequence corresponding to the coding region is written in one letter code.
canonical one, identified as AAUAAA, present in over 50% of plant mRNAs [22]. The lack of a strong consensus sequence may be a result of the presence of other regulatory mechanisms. In plant mRNAs there are cis-acting elements which enhance the polyadenylation signal. These elements are GU-rich [23]. Such motifs are present in the lupin 3% UTR of cDNA clone.
Northern analysis was done in order to confirm the size of expressed mRNA. As a result, one band (Fig. 2) of a length sufficient for coding a protein of 322 residues was detected. This result is consistent with that, obtained for R. nor6egicus ribosomal protein P0 gene organisation [6]. As we know, such analyses have not been done for other eukaryotic P0 proteins.
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Southern analysis of lupin genomic DNA carried out with restriction enzymes: BamHI, HindIII and EcoRI revealed six major bands (Fig. 3, lanes 1, 2) and five bands (Fig. 3, lane 3), respectively. The presence of more than one hybrydizing genomic DNA fragment would indicate that several P0 genes may exist in the lupin genome. Among eukaryotic organisms, P0 genes organization is known only for R. nor6egicus [6]. It revealed several copies of the gene in rat genome. Analyses of other ribosomal protein genes organisation in plants have shown the ex-
Fig. 2. Northern analysis of L. luteus total RNA. A band reflecting hybridization of the radiolabelled cDNA fragment (coding for the lupin ribosomal protein P0) to the lupin RNA, transferred to the nylon Hybond H + membrane as described by Feinberg and Vogelstein (1982) [18]. The positions of molecular length markers in bp are indicated.
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istence of family of genes for each protein, leading to the speculation that plants may have functionally distinct subsets of cytosolic ribosomes [24]. At present, in addition to the lupin P0 protein cDNA, three other cDNA sequences coding for plant ribosomal P0 proteins are known from: C. rubrum [25], O. sati6a [26], and G. max. We aligned them to other eukaryotic proteins P0 sequences (Fig. 4). We also included an EST of G. max protein P0 cDNA sequence (Dr J.A. Rafalski, personal communication). Surprisingly, we found that only plant P0 proteins contain specific motif RGD, which is not present in the other P0 proteins. Up to now this motif has been found in proteins involved in cell adhesion reactions ([27] and references therein). Generally, it is recognized by integrins which are gycoprotein receptors that mediate cell–cell and extracellular matrix–cell adhesion processes. It has been postulated that binding of RGD sequence to integrin might be responsible for high affinity ligand–acceptor interactions [28] and is also recognized by small GTP binding proteins involved in protein kinases regulation [29]. A question arises about the role to be played by RGD sequence in the ribosomal P0 protein. We think this region may be specific for plant ribosomal proteins P0 and could be involved in ribosome-cytoskeleton binding. The amino acid sequence of the yellow lupin ribosomal protein P0, deduced from cDNA, was compared to other P0s available in SwissProt Data Library [30]. This protein appeared to be homologous to higher eukaryotes P0 proteins and shows from 44% identity to the D. discoideum, to and 84% identity to the G. max. The alignment revealed that the lupin protein has conserved regions which are specific for P0 proteins from the other organisms which may be important for the interactions with rRNA and also with other ribosomal proteins [8,31,32]. In the C-terminal region there is a sequence: EEEPAEESDDE. Serine residue localized in this region is phosphorylated [33]. The C-terminal MGFGLFD sequence is highly con-
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Fig. 3. Southern analysis of L. luteus genomic DNA. Bands reflecting hybridization of radiolabelled cDNA (coding for L1P0) to the DNA digested with BamHI (1), HindIII (2), EcoRI(3) and transferred to the nylon Hybond H + membrane as described in Wu et al. (1995) [22]. The positions of molecular length markers in bp are indicated.
served among mammalian and yeast proteins P0 and P2. It is recognized by the antibodies present in the sera of patients with autoimmunological disease systemic lupus erythematosus [12]. This motif is also present in the lupin protein P0, which is not the case of other plant sequences (Fig. 4). The phylogenetic tree generated on the base of ribosomal proteins P0 (L10) sequences (Fig. 5) reflects phylogenetic distances between these proteins from different organisms, showing their molecular evolution. The attempts to find a correct tree using the distance matrix analysis re-
sulted in one diagram, shown in Fig. 5.
Acknowledgements We would like to thank Prof. A.B. Legocki and Dr M. Sikorski of this Institute for kindly providing us L. luteus cDNA library and Dr A. Rafalski of E.I. DuPont, Wilmington, DE., USA, for giving us G. max ESTs. This work was supported within the Committee of Scientific Research (KBN) grant.
Fig. 4. Alignment of aminoacid sequences of ribosomal proteins P0 from eukaryotic organisms; Dictyostelium discoideum, Trypanosoma cruzi, Leischmania chagasi, Saccharomyces cere6isiae (yeast), Chenopodium rubrum, Lupinus luteus (yellow lupin), Glycine max (soybean), Oryza sati6a (rice), Mus musculus (mouse), Rattus nor6egicus (rat), Homo sapiens (human being). The RGD sequence which is specific for plant P0 protein is in bold.
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Fig. 4. (continued)
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Fig. 5. The dendrogram, showing the relationships between the ribosomal proteins P0 sequences derived from protozoa (T. cruzi, L. chagasi ), yeast (S. cere6isae), higher plants (C. rubrum, O. sati6a, G. max, L. Luteus) and mammals (H. sapiens, R. nor6egicus, M. musculus). The numbers indicate percent confirmation of grouping of species to the right of the node by bootstraping analysis with 100 replications.
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